Substituted 2&#39;-thio-bicyclic nucleosides and oligomeric compounds prepared therefrom

ABSTRACT

Provided herein are novel bicyclic nucleosides, oligomeric compounds that include such bicyclic nucleosides and methods of using the oligomeric compounds. More particularly, the novel bicyclic nucleosides comprise a furanose ring system having a bridge comprising a 4′-methylene group attached to a 2′-sulfoxide or sulfone group and optionally including one or more substituent groups attached to the 4′-methylene and or the 5′-position. In certain embodiments, the oligomeric compounds provided herein are expected to hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/714,307, filed Oct. 16, 2012,which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCHEM0060USSEQ.TXT, created Oct. 7, 2012, which is 8 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are novel bicyclic nucleosides, oligomeric compoundsthat include such bicyclic nucleosides and methods of using theoligomeric compounds. More particularly, the novel bicyclic nucleosidescomprise a furanose ring system having a bridge comprising an optionallysubstituted 4′-methylene group attached to a 2′-sulfoxide or sulfonegroup and optionally including one or more substituent groups attachedto the 5′-position. In certain embodiments, the oligomeric compoundsprovided herein are expected to hybridize to a portion of a target RNAresulting in loss of normal function of the target RNA. The oligomericcompounds provided herein are also expected to be useful as primers andprobes in diagnostic applications.

BACKGROUND OF THE INVENTION

Targeting disease-causing gene sequences was first suggested more thanthirty years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), andantisense activity was demonstrated in cell culture more than a decadelater (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75,280-284). One advantage of antisense technology in the treatment of adisease or condition that stems from a disease-causing gene is that itis a direct genetic approach that has the ability to modulate (increaseor decrease) the expression of specific disease-causing genes. Anotheradvantage is that validation of a therapeutic target using antisensecompounds results in direct and immediate discovery of the drugcandidate; the antisense compound is the potential therapeutic agent.

Generally, the principle behind antisense technology is that anantisense compound hybridizes to a target nucleic acid and modulatesgene expression activities or function, such as transcription ortranslation. The modulation of gene expression can be achieved by, forexample, target degradation or occupancy-based inhibition. An example ofmodulation of RNA target function by degradation is RNase H-baseddegradation of the target RNA upon hybridization with a DNA-likeantisense compound. Another example of modulation of gene expression bytarget degradation is RNA interference (RNAi). RNAi generally refers toantisense-mediated gene silencing involving the introduction of dsRNAleading to the sequence-specific reduction of targeted endogenous mRNAlevels. Regardless of the specific mechanism, this sequence-specificitymakes antisense compounds extremely attractive as tools for targetvalidation and gene functionalization, as well as therapeutics toselectively modulate the expression of genes involved in thepathogenesis of malignancies and other diseases.

Antisense technology is an effective means for reducing the expressionof one or more specific gene products and can therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications. Chemically modified nucleosides are routinely used forincorporation into antisense compounds to enhance one or moreproperties, such as nuclease resistance, pharmacokinetics or affinityfor a target RNA. In 1998, the antisense compound, Vitravene®(fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.)was the first antisense drug to achieve marketing clearance from theU.S. Food and Drug Administration (FDA), and is currently a treatment ofcytomegalovirus (CMV)-induced retinitis in AIDS patients.

New chemical modifications have improved the potency and efficacy ofantisense compounds, uncovering the potential for oral delivery as wellas enhancing subcutaneous administration, decreasing potential for sideeffects, and leading to improvements in patient convenience. Chemicalmodifications increasing potency of antisense compounds allowadministration of lower doses, which reduces the potential for toxicity,as well as decreasing overall cost of therapy. Modifications increasingthe resistance to degradation result in slower clearance from the body,allowing for less frequent dosing. Different types of chemicalmodifications can be combined in one compound to further optimize thecompound's efficacy. One such group of chemical modifications includesbicyclic nucleosides wherein the furanose portion of the nucleosideincludes a bridge connecting two atoms on the furanose ring therebyforming a bicyclic ring system. Such bicyclic nucleosides have variousnames including BNA's and LNA's for bicyclic nucleic acids or lockednucleic acids respectively.

Various bicyclic nucleosides have been prepared and reported in thepatent literature as well as in scientific literature, see for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8(16), 2219-2222; Wengel et al., PCT InternationalApplication number PCT/DK98/00393 (published as WO 99/14226 on Mar. 25,1999), filed Sep. 14, 1998; Singh et al., J. Org. Chem., 1998, 63,10035-10039, the text of each is incorporated by reference herein, intheir entirety. Examples of issued US patents and published applicationsinclude for example: U.S. Pat. Nos. 6,770,748, 6,268,490 and 6,794,499and published U.S. applications 20040219565, 20040014959, 20030207841,20040192918, 20030224377, 20040143114, 20030087230 and 20030082807, thetext of each is incorporated by reference herein, in their entirety.

The synthesis of 5′-substituted DNA and RNA derivatives and theirincorporation into oligomeric compounds has been reported in theliterature (see for example: Saha et al., J. Org. Chem., 1995, 60,788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9,885-890; and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3),339-343) and Leonid et al., 1995, 14(3-5), 901-905).

The synthesis of 2′-thio bicyclic nucleosides has been reported in theliterature (see for example: International Application PCT/DK98/00393ibid; International Application PCT/DK-2004/000097, filed Feb. 10, 2004,and published as WO 2004/069992 on Aug. 19, 2004; Singh et al., Journalof Organic Chemistry, 1998, 63(18), 6078-6079; Pedersen et al.,Synthesis, 2004, 4, 578-582; U.S. Application 20040014959, publishedJan. 22, 2004; and U.S. Application 2004241717, published Dec. 2, 2004).

The synthesis of 2′-Thio bicyclic nucleosides and their incorporationinto oligomeric compounds has been reported in the literature. Selectedoligonucleotides have been looked at for evaluation of Tm, in vitroactivity and in vivo activity (see for example: Kumar ibid; and Fluiteret al., ChemBioChem, 2005, 6, 1-6).

There remains a long-felt need for agents that specifically regulategene expression via antisense mechanisms. Disclosed herein areoligomeric compounds such as antisense compounds useful for modulatinggene expression pathways, including those relying on mechanisms ofaction such as RNaseH, RNAi and dsRNA enzymes, as well as otherantisense mechanisms based on target degradation or target occupancy.One having skill in the art, once armed with this disclosure will beable, without undue experimentation, to identify, prepare and exploitantisense compounds for these uses.

BRIEF SUMMARY OF THE INVENTION

Provided herein are novel bicyclic nucleosides, oligomeric compoundsthat include such bicyclic nucleosides and methods of using theoligomeric compounds. More particularly, the novel bicyclic nucleosidescomprise a furanose ring system having a bridge comprising an optionallysubstituted 4′-methylene group attached to a 2′-sulfoxide or sulfonegroup and optionally including one or more substituent groups attachedto the 5′-position. The variables are defined individually in furtherdetail herein. It is to be understood that the substituted 2′-thiobicyclic nucleosides and oligomeric compounds provided herein includeall combinations of the embodiments disclosed and variables definedherein.

In certain embodiments, bicyclic nucleosides are provided having FormulaIa:

wherein:

Bx is a heterocyclic base moiety;

one of T₁ and T₂ is H or a hydroxyl protecting group and the other of T₁and T₂ is H, a hydroxyl protecting group or a reactive phosphorus group;

Q₁ and Q₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G₁ and G₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

each substituted group is, independently, mono or poly substituted withsubstituent groups independently selected from halogen, OJ₁, SJ₁, NJ₁J₂,N₃, CN, C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ andN(H)C(═S)NJ₁J₂;

each J₁ and J₂ is, independently, H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ aminoalkyl or a protecting group; and

n is 1 or 2.

In certain embodiments, Bx is an optionally protected pyrimidine,substituted pyrimidine, purine or substituted purine. In certainembodiments, Bx is uracil, thymine, cytosine, 4-N-benzoylcytosine,5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine

In certain embodiments, at least one of Q₁, Q₂, G₁ and G₂ is CH₃ and theremaining of Q₁, Q₂, G₁ and G₂ are each H. In certain embodiments, Q₁,Q₂, G₁ and G₂ are each H.

In certain embodiments, T₁ is 4,4′-dimethoxytrityl and T₂ isdiisopropylcyanoethoxy phosphoramidite.

In certain embodiments, n is 1 and the configuration at the sulfur atomis R. In certain embodiments, n is 1 and the configuration at the sulfuratom is S. In certain embodiments, n is 2.

In certain embodiments, Q₁, Q₂, G₁ and G₂ are each H, T₁ is4,4′-dimethoxytrityl and T₂ is diisopropylcyanoethoxy phosphoramidite.

In certain embodiments, oligomeric compounds are provided comprising atleast one bicyclic nucleoside of Formula IIa:

wherein independently for each bicyclic nucleoside of Formula IIa:

Bx is a heterocyclic base moiety;

one of T₃ and T₄ is an internucleoside linking group attaching thebicyclic nucleoside to the remainder of one of the 5′ or 3′ end of theoligomeric compound and the other of T₃ and T₄ is hydroxyl, a protectedhydroxyl, a 5′ or 3′ terminal group or an internucleoside linking groupattaching the bicyclic nucleoside to the remainder of the other of the5′ or 3′ end of the oligomeric compound;

Q₁ and Q₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G₁ and G₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

each substituted group is, independently, mono or poly substituted withsubstituent groups independently selected from halogen, OJ₁, SJ₁, NJ₁J₂,N₃, CN, C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ andN(H)C(═S)NJ₁J₂;

each J₁ and J₂ is, independently, H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ aminoalkyl or a protecting group;

n is 1 or 2; and

wherein said oligomeric compound comprises from 8 to 40 monomericsubunits linked by internucleoside linking groups and wherein at leastsome of the heterocyclic base moieties are capable of hybridizing to anucleic acid molecule.

In certain embodiments, each Bx is, independently, uracil, thymine,cytosine, 4-N-benzoylcytosine, 5-methylcytosine,4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine or2-N-isobutyrylguanine. In certain embodiments, each Bx is,independently, a purine, substituted purine, pyrimidine or substitutedpyrimidine.

In certain embodiments, at least one 5′ or 3′-conjugate group.

In certain embodiments, at least one of Q₁, Q₂, G₁ and G₂ is CH₃ and theremaining of Q₁,

Q₂, G₁ and G₂ are each H for each bicyclic nucleoside of Formula IIa. Incertain embodiments, Q₁,

Q₂, G₁ and G₂ are each H for each is bicyclic nucleoside of Formula IIa.

In certain embodiments, n is 1 and the configuration at the sulfur atomis R for each bicyclic nucleoside of Formula IIa. In certainembodiments, n is 1 and the configuration at the sulfur atom is S foreach bicyclic nucleoside of Formula IIa. In certain embodiments, n is 2for each bicyclic nucleoside of Formula IIa.

In certain embodiments, oligomeric compounds are provided comprisingfrom about 8 to about 20 linked monomer subunits.

In certain embodiments, gapped oligomeric compounds are providedcomprising a first region consisting of from 2 to 5 modifiednucleosides, a second region consisting of from 2 to 5 modifiednucleosides and a gap region located between the first and secondregions, wherein at least one of the modified nucleosides of the firstand second region is a bicyclic nucleoside having Formula IIa andwherein each monomer subunit in the gap region is independently, anucleoside or a modified nucleoside that is different from each bicyclicnucleoside of Formula IIa and each of the modified nucleosides in thefirst and second regions.

In certain embodiments, the gap region comprises from about 8 to about14 contiguous β-D-2′-deoxyribonucleosides.

In certain embodiments, essentially each modified nucleoside of thefirst and second region is a bicyclic nucleoside having Formula IIa. Incertain embodiments, each of the modified nucleosides that is other thana bicyclic nucleoside having Formula II is, independently, a substitutednucleoside having one or more sugar substituent groups, an optionallysubstituted 4′-S nucleoside or an optionally substituted bicyclicnucleoside.

In certain embodiments, each internucleoside linking group is,independently, a phosphodiester internucleoside linking group or aphosphorothioate internucleoside linking group. In certain embodiments,essentially each internucleoside linking group is a phosphorothioateinternucleoside linking group.

In certain embodiments, methods of inhibiting gene expression areprovided comprising contacting a cell with an oligomeric compound asprovided herein wherein said oligomeric compound is complementary to atarget RNA.

In certain embodiments, bicyclic nucleosides are provided herein havingFormula I:

wherein:

Bx is a heterocyclic base moiety;

one of T₁ and T₂ is H or a hydroxyl protecting group and the other of T₁and T₂ is H, a hydroxyl protecting group or a reactive phosphorus group;

Q₁ and Q₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G₁ and G₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

each substituted group is, independently, mono or poly substituted withsubstituent groups independently selected from halogen, OJ₁, SJ₁, NJ₁J₂,N₃, CN, C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ andN(H)C(═S)NJ₁J₂;

each J₁ and J₂ is, independently, H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ aminoalkyl or a protecting group; and

n is 1 or 2. In certain embodiments, Bx is an optionally protectedpyrimidine, substituted pyrimidine, purine or substituted purine. Incertain embodiments, Bx is uracil, thymine, cytosine,4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine,adenine, 6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine

In certain embodiments, T₁ is H or a hydroxyl protecting group. Incertain embodiments, T₂ is a reactive phosphorus group selected from anH-phosphonate or a phosphoramidite. In certain embodiments, T₁ is4,4′-dimethoxytrityl. In certain embodiments, T₁ is 4,4′-dimethoxytrityland T₂ is diisopropylcyanoethoxy phosphoramidite.

In certain embodiments, one of Q₁ and Q₂ is H and the other of Q₁ and Q₂is other than H. In certain embodiments, at least one of Q₁ and Q₂ isC₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certain embodiments, at leastone of Q₁ and Q₂ is methyl. In certain embodiments, Q₁ and Q₂ are eachH.

In certain embodiments, one of G₁ and G₂ is H and the other of G₁ and Gis other than H. In certain embodiments, at least one of G₁ and G₂ isC₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certain embodiments, at leastone of G₁ and G₂ is methyl. In certain embodiments, G₁ and G₂ are eachH. In certain embodiments, Q₁, Q₂, G₁ and G₂ are each H.

In certain embodiments, n is 1. In certain embodiments, n is 2.

In certain embodiments, the bicyclic nucleoside having Formula I has theconfiguration of Formula Ia:

In certain embodiments, three of Q₁, Q₂, G₁ and G₂ are H and the otherone of Q₁, Q₂, G₁ and G₂ is other than H. In certain embodiments, one ofQ₁, Q₂, G₁ and G₂ is CH₃.

In certain embodiments, n is 1 and the configuration at the sulfur atomis R. In certain embodiments, n is 1 and the configuration at the sulfuratom is S. In certain embodiments, n is 2.

In certain embodiments, oligomeric compounds are provided comprising atleast one bicyclic nucleoside of Formula II:

wherein independently for each bicyclic nucleoside of Formula II:

Bx is a heterocyclic base moiety;

one of T₃ and T₄ is an internucleoside linking group attaching thebicyclic nucleoside to the remainder of one of the 5′ or 3′ end of theoligomeric compound and the other of T₃ and T₄ is hydroxyl, a protectedhydroxyl, a 5′ or 3′ terminal group or an internucleoside linking groupattaching the bicyclic nucleoside to the remainder of the other of the5′ or 3′ end of the oligomeric compound;

Q₁ and Q₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G₁ and G₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

each substituted group is, independently, mono or poly substituted withsubstituent groups independently selected from halogen, OJ₁, SJ₁, NJ₁J₂,N₃, CN, C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ andN(H)C(═S)NJ₁J₂;

each J₁ and J₂ is, independently, H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ aminoalkyl or a protecting group;

n is 1 or 2; and

wherein the oligomeric compound comprises from 8 to 40 monomericsubunits linked by internucleoside linking groups and wherein at leastsome of the heterocyclic base moieties are capable of hybridizing to anucleic acid molecule.

In certain embodiments, n each Bx is, independently, an optionallyprotected pyrimidine, substituted pyrimidine, purine or substitutedpurine. In certain embodiments, each Bx is, independently, uracil,thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine,4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine or2-N-isobutyrylguanine

In certain embodiments, at least one of T₃ and T₄ is a 5′ or 3′-terminalgroup. In certain embodiments, at least one 5′ or 3′-terminal group is aconjugate group. In certain embodiments, at least one 5′ or 3′-terminalgroup is a phosphate moiety.

In certain embodiments, one of Q₁ and Q₂ is H and the other of Q₁ and Q₂is other than H for each bicyclic nucleoside of Formula II. In certainembodiments, one of Q₁ and Q₂ is C₁-C₆ alkyl or substituted C₁-C₆ alkylfor each bicyclic nucleoside of Formula II. In certain embodiments, oneof Q₁ and Q₂ is methyl for each bicyclic nucleoside of Formula II. Incertain embodiments, one of Q₁ and Q₂ are each H for each bicyclicnucleoside of Formula II.

In certain embodiments, one of G₁ and G₂ is H and the other of G₁ and G₂is other than H for each bicyclic nucleoside of Formula II. In certainembodiments, one of G₁ and G₂ is C₁-C₆ alkyl or substituted C₁-C₆ alkylfor each bicyclic nucleoside of Formula II. In certain embodiments, oneof G₁ and G₂ is methyl for each bicyclic nucleoside of Formula II. Incertain embodiments, G₁ and G₂ are each H for each bicyclic nucleosideof Formula II. In certain embodiments, Q₁, Q₂, G₁ and G₂ are each H foreach bicyclic nucleoside of Formula II.

In certain embodiments, n is 1 for each bicyclic nucleoside of FormulaII. In certain embodiments, n is 2 for each bicyclic nucleoside ofFormula II.

In certain embodiments, oligomeric compounds are provided comprising atleast one bicyclic nucleoside of Formula II that each have theconfiguration of Formula IIa:

In certain embodiments, three of Q₁, Q₂, G₁ and G₂ are H and the otherone of Q₁, Q₂, G₁ and G₂ is other than H for each bicyclic nucleoside ofFormula IIa. In certain embodiments, one of Q₁, Q₂, G₁ and G₂ is methylfor each bicyclic nucleoside of Formula IIa. In certain embodiments, Q₁,Q₂, G₁ and G₂ are each H for each bicyclic nucleoside of Formula IIa.

In certain embodiments, n is 1 and the configuration at the sulfur atomis R for each bicyclic nucleoside of Formula IIa. In certainembodiments, n is 1 and the configuration at the sulfur atom is S foreach bicyclic nucleoside of Formula IIa. In certain embodiments, n is 2for each bicyclic nucleoside of Formula IIa.

In certain embodiments, oligomeric compounds are provided comprisingfrom about 8 to about 20 linked monomer subunits. In certainembodiments, oligomeric compounds are provided comprising from about 10to about 18 linked monomer subunits. In certain embodiments, oligomericcompounds are provided comprising from about 12 to about 16 linkedmonomer subunits.

In certain embodiments, oligomeric compounds are provided comprising afirst region consisting of from 2 to 5 modified nucleosides, wherein atleast one of the modified nucleosides of the first region is a bicyclicnucleoside having Formula II. In certain embodiments, oligomericcompounds are provided comprising a first region consisting of from 2 to5 modified nucleosides and a second region consisting of from two to 5modified nucleosides wherein at least one of the modified nucleosides ofthe first region is a bicyclic nucleoside having Formula II. In certainembodiments, oligomeric compounds are provided comprising a first regionconsisting of from 2 to 5 modified nucleosides and a second regionconsisting of from two to 5 modified nucleosides wherein at least one ofthe modified nucleosides of the first region is a bicyclic nucleosidehaving Formula II and at least one of the modified nucleosides of thesecond region is a bicyclic nucleoside having Formula II.

In certain embodiments, oligomeric compounds are provided comprising agap region located between the first and second regions wherein eachmonomer subunit in the gap region is independently, a nucleoside or amodified nucleoside that is different from each bicyclic nucleoside ofFormula II and each of the modified nucleosides in the first and secondregions. In certain embodiments, the gap region comprises from about 6to about 14 monomer subunits. In certain embodiments, the gap regioncomprises from about 8 to about 14 contiguousβ-D-2′-deoxyribonucleosides. In certain embodiments, the gap regioncomprises from about 9 to about 12 contiguousβ-D-2′-deoxyribonucleosides. In certain embodiments, essentially eachmodified nucleoside of the first region is a bicyclic nucleoside havingFormula II. In certain embodiments, essentially each modified nucleosideof the first and second regions is a bicyclic nucleoside having FormulaII.

In certain embodiments, oligomeric compounds are provided wherein eachof the modified nucleosides that is other than a bicyclic nucleosidehaving Formula II is, independently, a substituted nucleoside having oneor more sugar substituent groups, an optionally substituted 4′-Snucleoside or an optionally substituted bicyclic nucleoside. In certainembodiments, each of the modified nucleosides that is other than abicyclic nucleoside having Formula II is, independently, a 2′substituted nucleoside comprising a 2′-fluoro, 2′-O—CH₃,2′-O—(CH₂)₂—O—CH₃ or 2′-O—CH₂C(═O)—N(H)(CH₃) sugar substituent group. Incertain embodiments, each of the modified nucleosides that is other thana bicyclic nucleoside having Formula II is a 2′ substituted nucleosidecomprising a 2′-O—(CH₂)₂—O—CH₃ sugar substituent group.

In certain embodiments, oligomeric compounds are provided wherein eachbicyclic nucleoside of Formula II has the configuration of Formula IIa.

In certain embodiments, oligomeric compounds are provided wherein eachinternucleoside linking group is, independently, a phosphodiesterinternucleoside linking group or a phosphorothioate internucleosidelinking group. In certain embodiments, essentially each internucleosidelinking group is a phosphorothioate internucleoside linking group.

In certain embodiments, methods of inhibiting gene expression areprovided comprising contacting a cell with an oligomeric compound of anyof claims 25 to 65 wherein the oligomeric compound is complementary to atarget RNA. In certain embodiments, the cell is in an animal. In certainembodiments, the cell is in a human. In certain embodiments, the targetRNA is selected from mRNA, pre-mRNA and micro RNA. In certainembodiments, the target RNA is mRNA. In certain embodiments, the targetRNA is human mRNA. In certain embodiments, the target RNA is cleavedthereby inhibiting its function. In certain embodiments, the methodsfurther comprise detecting the levels of target RNA.

In certain embodiments, methods of inhibiting gene expression areprovided comprising contacting one or more cells or a tissue with anoligomeric compound of any of claims 25 to 66.

In certain embodiments, oligomeric compounds are provided fortherapeutic use in an in vivo method of inhibiting gene expression themethod comprising contacting an animal with an oligomeric compound asprovided herein.

In certain embodiments, oligomeric compounds are provided for use inmedical therapy.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are novel substituted 2′-thio bicyclic nucleosides andoligomeric compounds prepared therefrom. More particularly, thesubstituted 2′-thio bicyclic nucleosides provided herein include afuranose ring system having a bridge comprising an optionallysubstituted 4′-methylene group attached to a 2′-sulfoxide or sulfonegroup and optionally include one or more substituent groups attached tothe 5′-position. In certain embodiments, the oligomeric compoundsprovided herein are expected to hybridize to a portion of a target RNAresulting in loss of normal function of the target RNA.

Incorporation of one or more of the substituted 2′-thio bicyclicnucleosides, as provided herein, into an oligomeric compound is expectedto enhance one or more desired properties of the resulting oligomericcompound. Such properties include without limitation stability, nucleaseresistance, binding affinity, specificity, absorption, cellulardistribution, cellular uptake, charge, pharmacodynamics andpharmacokinetics.

In certain embodiments, the substituted 2′-thio bicyclic nucleosidesprovided herein are incorporated into antisense oligomeric compoundswhich are used to reduce target RNA, such as messenger RNA, in vitro andin vivo. The reduction of target RNA can be effected via numerouspathways with a resultant modulation of gene expression. Such modulationcan provide direct or indirect increase or decrease in a particulartarget (nucleic acid or protein). Such pathways include for example thesteric blocking of transcription or translation and cleavage of mRNAusing either single or double stranded oligomeric compounds. Theoligomeric compounds provided herein are also expected to be useful asprimers and probes in diagnostic applications. In certain embodiments,oligomeric compounds comprising at least one of the substituted 2′-thiobicyclic nucleosides provided herein are expected to be useful asaptamers which are oligomeric compounds capable of binding to aberrantproteins in an in vivo setting.

In certain embodiments, the substituted 2′-thio bicyclic nucleosidesprovided herein each have Formula I:

wherein:

Bx is a heterocyclic base moiety;

one of T₁ and T₂ is H or a hydroxyl protecting group and the other of T₁and T₂ is H, a hydroxyl protecting group or a reactive phosphorus group;

Q₁ and Q₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G₁ and G₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

each substituted group is, independently, mono or poly substituted withsubstituent groups independently selected from halogen, OJ₁, SJ₁, NJ₁J₂,N₃, CN, C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ andN(H)C(═S)NJ₁J₂;

each J₁ and J₂ is, independently, H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ aminoalkyl or a protecting group; and

n is 1 or 2.

In certain embodiments, each of the substituted 2′-thio bicyclicnucleosides having Formula I further have the configuration of FormulaIa:

In certain embodiments, oligomeric compounds are provided comprising atleast one bicyclic nucleoside of Formula II:

wherein independently for each bicyclic nucleoside of Formula II:

Bx is a heterocyclic base moiety;

one of T₃ and T₄ is an internucleoside linking group attaching thebicyclic nucleoside to the remainder of one of the 5′ or 3′ end of theoligomeric compound and the other of T₃ and T₄ is hydroxyl, a protectedhydroxyl, a 5′ or 3′ terminal group or an internucleoside linking groupattaching the bicyclic nucleoside to the remainder of the other of the5′ or 3′ end of the oligomeric compound;

Q₁ and Q₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G₁ and G₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

each substituted group is, independently, mono or poly substituted withsubstituent groups independently selected from halogen, OJ₁, SJ₁, NJ₁J₂,N₃, CN, C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ andN(H)C(═S)NJ₁J₂;

each J₁ and J₂ is, independently, H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ aminoalkyl or a protecting group;

n is 1 or 2; and

wherein said oligomeric compound comprises from 8 to 40 monomericsubunits linked by internucleoside linking groups and wherein at leastsome of the heterocyclic base moieties are capable of hybridizing to anucleic acid molecule.

In certain embodiments, oligomeric compounds are provided comprising atleast one bicyclic nucleoside of Formula II wherein each bicyclicnucleoside of Formula II further has the configuration of Formula IIa:

In certain embodiments, the substituted 2′-thio bicyclic nucleosidesprovided herein are incorporated into oligomeric compounds such that amotif results. The placement of substituted 2′-thio bicyclic nucleosidesinto oligomeric compounds to provide particular motifs can enhance thedesired properties of the resulting oligomeric compounds for activityusing various mechanisms such as for example RNaseH or RNAi. Such motifsinclude without limitation, gapmer motifs, hemimer motifs, blockmermotifs, uniformly fully modified motifs, positionally modified motifsand alternating motifs. In conjunction with these motifs a wide varietyof internucleoside linkages can also be used including but not limitedto phosphodiester and phosphorothioate internucleoside linkages whichcan be incorporated uniformly or in various combinations. The oligomericcompounds can further include terminal groups at one or both of the 5′and or 3′ terminals such as a conjugate or reporter group. Thepositioning of the substituted 2′-thio bicyclic nucleosides providedherein, the use of linkage strategies and terminal groups can be easilyoptimized to enhance a desired activity for a selected target.

As used herein the term “motif” refers to the pattern created by therelative positioning of monomer subunits within an oligomeric compoundwherein the pattern is determined by comparing the sugar moieties of thelinked monomer subunits. The only determinant for the motif of anoligomeric compound is the differences or lack of differences betweenthe sugar moieties. The internucleoside linkages, heterocyclic bases andfurther groups such as terminal groups are not considered whendetermining the motif of an oligomeric compound.

The preparation of motifs has been disclosed in various publicationsincluding without limitation, representative U.S. Pat. Nos. 5,013,830;5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133;5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922; and publishedinternational applications WO 2005/121371 and WO 2005/121372 (bothpublished on Dec. 22, 2005), certain of which are commonly owned withthe instant application, and each of which is herein incorporated byreference in its entirety.

As used herein the term “alternating motif” refers to an oligomericcompound comprising a contiguous sequence of linked monomer subunitswherein the monomer subunits have two different types of sugar moietiesthat alternate for essentially the entire sequence of the oligomericcompound. Oligomeric compounds having an alternating motif can bedescribed by the formula: 5′-A(-L-B-L-A)_(n)(-L-B)_(nn)-3′ where A and Bare monomer subunits that have different sugar moieties, each L is,independently, an internucleoside linking group, n is from about 4 toabout 12 and nn is 0 or 1. The heterocyclic base and internucleosidelinkage is independently variable at each position. The motif furtheroptionally includes the use of one or more other groups including butnot limited to capping groups, conjugate groups and other 5′ and or3′-terminal groups. This permits alternating oligomeric compounds fromabout 9 to about 26 monomer subunits in length. This length range is notmeant to be limiting as longer and shorter oligomeric compounds are alsoamenable to oligomeric compounds provided herein. In certainembodiments, each A or each B comprise 2′-thio bicyclic nucleosides asprovided herein.

As used herein the term “uniformly fully modified motif” refers to anoligomeric compound comprising a contiguous sequence of linked monomersubunits that each have the same type of sugar moiety. The heterocyclicbase and internucleoside linkage is independently variable at eachposition. The motif further optionally includes the use of one or moreother groups including but not limited to capping groups, conjugategroups and other 5′ and or 3′-terminal groups. In certain embodiments,the uniformly fully modified motif includes a contiguous sequence of2′-thio bicyclic nucleosides. In certain embodiments, one or both of the5′ and 3′-ends of the contiguous sequence of 2′-thio bicyclicnucleosides, comprise 5′ and or 3′-terminal groups such as one or moreunmodified nucleosides.

As used herein the term “hemimer motif” refers to an oligomeric compoundcomprising a contiguous sequence of monomer subunits that each have thesame type of sugar moiety with a further short contiguous sequence ofmonomer subunits located at the 5′ or the 3′ end that have a differenttype of sugar moiety. The heterocyclic base and internucleoside linkageis independently variable at each position. The motif further optionallyincludes the use of one or more other groups including but not limitedto capping groups, conjugate groups and other 5′ and or 3′-terminalgroups. In general, a hemimer is an oligomeric compound of uniform sugarmoieties further comprising a short region (1, 2, 3, 4 or about 5monomer subunits) having uniform but different sugar moieties located oneither the 3′ or the 5′ end of the oligomeric compound.

In certain embodiments, the hemimer motif comprises a contiguoussequence of from about 10 to about 28 monomer subunits having one typeof sugar moiety with from 1 to 5 or from 2 to about 5 monomer subunitshaving a second type of sugar moiety located at one of the termini. Incertain embodiments, the hemimer is a contiguous sequence of from about8 to about 20 β-D-2′-deoxyribonucleosides having from 1-12 contiguous2′-thio bicyclic nucleosides located at one of the termini. In certainembodiments, the hemimer is a contiguous sequence of from about 8 toabout 20 β-D-2′-deoxyribonucleosides having from 1-5 contiguous 2′-thiobicyclic nucleosides located at one of the termini. In certainembodiments, the hemimer is a contiguous sequence of from about 12 toabout 18 β-D-2′-deoxyribonucleosides having from 1-3 contiguous 2′-thiobicyclic nucleosides located at one of the termini. In certainembodiments, the hemimer is a contiguous sequence of from about 10 toabout 14 β-D-2′-deoxyribonucleosides having from 1-3 contiguous 2′-thiobicyclic nucleosides located at one of the termini.

As used herein the terms “blockmer motif” and “blockmer” refer to anoligomeric compound comprising an otherwise contiguous sequence ofmonomer subunits wherein the sugar moieties of each monomer subunit isthe same except for an interrupting internal block of contiguous monomersubunits having a different type of sugar moiety. The heterocyclic baseand internucleoside linkage is independently variable at each positionof a blockmer. The motif further optionally includes the use of one ormore other groups including but not limited to capping groups, conjugategroups and other 5′ or 3′-terminal groups. A blockmer overlaps somewhatwith a gapmer in the definition but typically only the monomer subunitsin the block have non-naturally occurring sugar moieties in a blockmerand only the monomer subunits in the external regions have non-naturallyoccurring sugar moieties in a gapmer with the remainder of monomersubunits in the blockmer or gapmer being β-D-2′-deoxyribonucleosides orβ-D-ribonucleosides. In certain embodiments, blockmers are providedherein wherein all of the monomer subunits comprise non-naturallyoccurring sugar moieties.

As used herein the term “positionally modified motif” is meant toinclude an otherwise contiguous sequence of monomer subunits having onetype of sugar moiety that is interrupted with two or more regions offrom 1 to about 5 contiguous monomer subunits having another type ofsugar moiety. Each of the two or more regions of from 1 to about 5contiguous monomer subunits are independently uniformly modified withrespect to the type of sugar moiety. In certain embodiments, each of thetwo or more regions have the same type of sugar moiety. In certainembodiments, each of the two or more regions have a different type ofsugar moiety. In certain embodiments, each of the two or more regions,independently, have the same or a different type of sugar moiety. Theheterocyclic base and internucleoside linkage is independently variableat each position of a positionally modified oligomeric compound. Themotif further optionally includes the use of one or more other groupsincluding but not limited to capping groups, conjugate groups and other5′ or 3′-terminal groups. In certain embodiments, positionally modifiedoligomeric compounds are provided comprising a sequence of from 8 to 20β-D-2′-deoxyribonucleosides that further includes two or three regionsof from 2 to about 5 contiguous 2′-thio bicyclic nucleosides each.Positionally modified oligomeric compounds are distinguished from gappedmotifs, hemimer motifs, blockmer motifs and alternating motifs becausethe pattern of regional substitution defined by any positional motifdoes not fit into the definition provided herein for one of these othermotifs. The term positionally modified oligomeric compound includes manydifferent specific substitution patterns.

As used herein the term “gapmer” or “gapped oligomeric compound” refersto an oligomeric compound having two external regions or wings and aninternal region or gap. The three regions form a contiguous sequence ofmonomer subunits with the sugar moieties of the external regions beingdifferent than the sugar moieties of the internal region and wherein thesugar moiety of each monomer subunit within a particular region isessentially the same. In certain embodiments, each monomer subunitwithin a particular region has the same sugar moiety. When the sugarmoieties of the external regions are the same the gapmer is a symmetricgapmer and when the sugar moiety used in the 5′-external region isdifferent from the sugar moiety used in the 3′-external region, thegapmer is an asymmetric gapmer. In certain embodiments, the externalregions are small (each independently 1, 2, 3, 4 or about 5 monomersubunits) and the monomer subunits comprise non-naturally occurringsugar moieties with the internal region comprisingβ-D-2′-deoxyribonucleosides. In certain embodiments, the externalregions each, independently, comprise from 1 to about 5 monomer subunitshaving non-naturally occurring sugar moieties and the internal regioncomprises from 6 to 18 unmodified nucleosides. The internal region orthe gap generally comprises β-D-2′-deoxyribonucleosides but can comprisenon-naturally occurring sugar moieties. The heterocyclic base andinternucleoside linkage is independently variable at each position of agapped oligomeric compound. The motif further optionally includes theuse of one or more other groups including but not limited to cappinggroups, conjugate groups and other 5′ or 3′-terminal groups.

In certain embodiments, the gapped oligomeric compounds comprise aninternal region of β-D-2′-deoxyribonucleosides with one of the externalregions comprising 2′-thio bicyclic nucleosides as disclosed herein. Incertain embodiments, the gapped oligomeric compounds comprise aninternal region of β-D-2′-deoxyribonucleosides with one of the externalregions comprising 2′-thio bicyclic nucleosides as disclosed herein andthe other external region comprising modified nucleosides different thanthe 2′-thio bicyclic nucleosides as disclosed herein. In certainembodiments, the gapped oligomeric compounds comprise an internal regionof β-D-2′-deoxyribonucleosides with both of the external regionscomprising 2′-thio bicyclic nucleosides as provided herein. In certainembodiments, the gapped oligomeric compounds comprise an internal regionof β-D-2′-deoxyribonucleosides with both of the external regionscomprising a mixture of 2′-thio bicyclic nucleosides as provided hereinand at least one other modified nucleosides different from the 2′-thiobicyclic nucleosides as provided herein. In certain embodiments, gappedoligomeric compounds are provided herein wherein all of the monomersubunits comprise non-naturally occurring sugar moieties.

In certain embodiments, gapped oligomeric compounds are providedcomprising one or two 2′-thio bicyclic nucleosides at the 5′-end, two orthree 2′-thio bicyclic nucleosides at the 3′-end and an internal regionof from 10 to 16 β-D-2′-deoxyribonucleosides. In certain embodiments,gapped oligomeric compounds are provided comprising one of the 2′-thiobicyclic nucleosides at the 5′-end, two 2′-thio bicyclic nucleosides atthe 3′-end and an internal region of from 10 to 16β-D-2′-deoxyribonucleosides. In certain embodiments, gapped oligomericcompounds are provided comprising one 2′-thio bicyclic nucleosides atthe 5′-end, two 2′-thio bicyclic nucleosides at the 3′-end and aninternal region of from 10 to 14 β-D-2′-deoxyribonucleosides. In certainembodiments, gapped oligomeric compounds are provided comprising one ormore 2′-thio bicyclic nucleosides at the 5′-end, one or more 2′-thiobicyclic nucleosides at the 3′-end and an internal region of from 8 to14 β-D-2′-deoxyribonucleosides wherein each of the 3′-end and 5′-endfurther include from 1 to 3 modified nucleosides different from the2′-thio bicyclic nucleosides. In certain embodiments, gapped oligomericcompounds are provided comprising one or more 2′-thio bicyclicnucleosides at the 5′-end, one or more 2′-thio bicyclic nucleosides atthe 3′-end and an internal region of from 8 to 14β-D-2′-deoxyribonucleosides wherein each of the 3′-end and 5′-endfurther include from 1 to 3 MOE modified nucleosides.

In certain embodiments, gapped oligomeric compounds are provided thatare from about 18 to about 21 monomer subunits in length. In certainembodiments, gapped oligomeric compounds are provided that are fromabout 16 to about 21 monomer subunits in length. In certain embodiments,gapped oligomeric compounds are provided that are from about 10 to about21 monomer subunits in length. In certain embodiments, gapped oligomericcompounds are provided that are from about 12 to about 16 monomersubunits in length. In certain embodiments, gapped oligomeric compoundsare provided that are from about 12 to about 14 monomer subunits inlength. In certain embodiments, gapped oligomeric compounds are providedthat are from about 14 to about 16 monomer subunits in length.

As used herein the term “alkyl,” refers to a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred. The term “lower alkyl” asused herein includes from 1 to about 6 carbon atoms. Alkyl groups asused herein may optionally include one or more further substituentgroups.

As used herein the term “alkenyl,” refers to a straight or branchedhydrocarbon chain radical containing up to twenty four carbon atoms andhaving at least one carbon-carbon double bond. Examples of alkenylgroups include without limitation, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like.Alkenyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkenyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein the term “alkynyl,” refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms and havingat least one carbon-carbon triple bond. Examples of alkynyl groupsinclude, without limitation, ethynyl, 1-propynyl, 1-butynyl, and thelike. Alkynyl groups typically include from 2 to about 24 carbon atoms,more typically from 2 to about 12 carbon atoms with from 2 to about 6carbon atoms being more preferred. Alkynyl groups as used herein mayoptionally include one or more further substituent groups.

As used herein the term “aliphatic,” refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms whereinthe saturation between any two carbon atoms is a single, double ortriple bond. An aliphatic group preferably contains from 1 to about 24carbon atoms, more typically from 1 to about 12 carbon atoms with from 1to about 6 carbon atoms being more preferred. The straight or branchedchain of an aliphatic group may be interrupted with one or moreheteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Suchaliphatic groups interrupted by heteroatoms include without limitation,polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines.Aliphatic groups as used herein may optionally include furthersubstituent groups.

As used herein the term “alicyclic” refers to a cyclic ring systemwherein the ring is aliphatic. The ring system can comprise one or morerings wherein at least one ring is aliphatic. Preferred alicyclicsinclude rings having from about 5 to about 9 carbon atoms in the ring.Alicyclic as used herein may optionally include further substituentgroups.

As used herein the term “alkoxy,” refers to a radical formed between analkyl group and an oxygen atom wherein the oxygen atom is used to attachthe alkoxy group to a parent molecule. Examples of alkoxy groups includewithout limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like.Alkoxy groups as used herein may optionally include further substituentgroups.

As used herein the term “aminoalkyl” refers to an amino substitutedC₁-C₁₂ alkyl radical. The alkyl portion of the radical forms a covalentbond with a parent molecule. The amino group can be located at anyposition and the aminoalkyl group can be substituted with a furthersubstituent group at the alkyl and/or amino portions.

As used herein the terms “aryl” and “aromatic,” refer to a mono- orpolycyclic carbocyclic ring system radicals having one or more aromaticrings. Examples of aryl groups include without limitation, phenyl,naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferredaryl ring systems have from about 5 to about 20 carbon atoms in one ormore rings. Aryl groups as used herein may optionally include furthersubstituent groups.

As used herein the terms “aralkyl” and “arylalkyl,” refer to an aromaticgroup that is covalently linked to a C₁-C₁₂ alkyl radical. The alkylradical portion of the resulting aralkyl (or arylalkyl) group forms acovalent bond with a parent molecule. Examples include withoutlimitation, benzyl, phenethyl and the like. Aralkyl groups as usedherein may optionally include further substituent groups attached to thealkyl, the aryl or both groups that form the radical group.

As used herein the term “heterocyclic radical” refers to a radicalmono-, or poly-cyclic ring system that includes at least one heteroatomand is unsaturated, partially saturated or fully saturated, therebyincluding heteroaryl groups. Heterocyclic is also meant to include fusedring systems wherein one or more of the fused rings contain at least oneheteroatom and the other rings can contain one or more heteroatoms oroptionally contain no heteroatoms. A heterocyclic radical typicallyincludes at least one atom selected from sulfur, nitrogen or oxygen.Examples of heterocyclic radicals include, [1,3]dioxolanyl,pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuryl and the like. Heterocyclic groups as used herein mayoptionally include further substituent groups.

As used herein the terms “heteroaryl,” and “heteroaromatic,” refer to aradical comprising a mono- or poly-cyclic aromatic ring, ring system orfused ring system wherein at least one of the rings is aromatic andincludes one or more heteroatoms. Heteroaryl is also meant to includefused ring systems including systems where one or more of the fusedrings contain no heteroatoms. Heteroaryl groups typically include onering atom selected from sulfur, nitrogen or oxygen. Examples ofheteroaryl groups include without limitation, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like.Heteroaryl radicals can be attached to a parent molecule directly orthrough a linking moiety such as an aliphatic group or hetero atom.Heteroaryl groups as used herein may optionally include furthersubstituent groups.

As used herein the term “heteroarylalkyl,” refers to a heteroaryl groupas previously defined that further includes a covalently attached C₁-C₁₂alkyl radical. The alkyl radical portion of the resultingheteroarylalkyl group is capable of forming a covalent bond with aparent molecule. Examples include without limitation,pyridinylmethylene, pyrimidinylethylene, napthyridinylpropylene and thelike. Heteroarylalkyl groups as used herein may optionally includefurther substituent groups on one or both of the heteroaryl or alkylportions.

As used herein the term “acyl,” refers to a radical formed by removal ofa hydroxyl group from an organic acid and has the general Formula—C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examplesinclude aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls,aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein the term “hydrocarbyl” includes radical groups thatcomprise C, O and H. Included are straight, branched and cyclic groupshaving any degree of saturation. Such hydrocarbyl groups can include oneor more additional heteroatoms selected from N and S and can be furthermono or poly substituted with one or more substituent groups.

As used herein the term “mono or polycyclic ring system” is meant toinclude all ring systems selected from single or polycyclic radical ringsystems wherein the rings are fused or linked and is meant to beinclusive of single and mixed ring systems individually selected fromaliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl,heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such monoand poly cyclic structures can contain rings that each have the samelevel of saturation or each, independently, have varying degrees ofsaturation including fully saturated, partially saturated or fullyunsaturated. Each ring can comprise ring atoms selected from C, N, O andS to give rise to heterocyclic rings as well as rings comprising only Cring atoms which can be present in a mixed motif such as for examplebenzimidazole wherein one ring has only carbon ring atoms and the fusedring has two nitrogen atoms. The mono or polycyclic ring system can befurther substituted with substituent groups such as for examplephthalimide which has two ═O groups attached to one of the rings. Monoor polycyclic ring systems can be attached to parent molecules usingvarious strategies such as directly through a ring atom, fused throughmultiple ring atoms, through a substituent group or through abifunctional linking moiety.

As used herein the terms “halo” and “halogen,” refer to an atom selectedfrom fluorine, chlorine, bromine and iodine.

As used herein the term “oxo” refers to the group (═O).

As used herein the term “protecting group,” refers to a labile chemicalmoiety which is known in the art to protect reactive groups includingwithout limitation, hydroxyl, amino and thiol groups, against undesiredreactions during synthetic procedures. Protecting groups are typicallyused selectively and/or orthogonally to protect sites during reactionsat other reactive sites and can then be removed to leave the unprotectedgroup as is or available for further reactions. Protecting groups asknown in the art are described generally in Greene's Protective Groupsin Organic Synthesis, 4th edition, John Wiley & Sons, New York, 2007.

Groups can be selectively incorporated into oligomeric compounds asprovided herein as precursors. For example an amino group can be placedinto a compound as provided herein as an azido group that can bechemically converted to the amino group at a desired point in thesynthesis. Generally, groups are protected or present as precursors thatwill be inert to reactions that modify other areas of the parentmolecule for conversion into their final groups at an appropriate time.Further representative protecting or precursor groups are discussed inAgrawal et al., Protocols for Oligonucleotide Conjugates, Humana Press;New Jersey, 1994, 26, 1-72.

The term “orthogonally protected” refers to functional groups which areprotected with different classes of protecting groups, wherein eachclass of protecting group can be removed in any order and in thepresence of all other classes (see, Barany et al., J. Am. Chem. Soc.,1977, 99, 7363-7365; Barany et al., J. Am. Chem. Soc., 1980, 102,3084-3095). Orthogonal protection is widely used in for exampleautomated oligonucleotide synthesis. A functional group is deblocked inthe presence of one or more other protected functional groups which isnot affected by the deblocking procedure. This deblocked functionalgroup is reacted in some manner and at some point a further orthogonalprotecting group is removed under a different set of reactionconditions. This allows for selective chemistry to arrive at a desiredcompound or oligomeric compound.

Examples of hydroxyl protecting groups include without limitation,acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl(TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl,pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate,mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl,dimethoxytrityl (DMT), trimethoxytrityl,1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl(Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Wherein morecommonly used hydroxyl protecting groups include without limitation,benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,benzoyl, mesylate, tosylate, dimethoxytrityl (DMT),9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl(MOX).

Examples of amino protecting groups include without limitation,carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; andimine- and cyclic imide-protecting groups, such as phthalimido anddithiasuccinoyl.

Examples of thiol protecting groups include without limitation,triphenylmethyl (trityl), benzyl (Bn), and the like.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, α or β, or as (D)- or (L)- such as foramino acids. Included herein are all such possible isomers, as well astheir racemic and optically pure forms. Optical isomers may be preparedfrom their respective optically active precursors by the proceduresdescribed above, or by resolving the racemic mixtures. The resolutioncan be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions, John Wiley & Sons, 1981. Whenthe compounds described herein contain olefinic double bonds, otherunsaturation, or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both Eand Z geometric isomers or cis- and trans-isomers. Likewise, alltautomeric forms are also intended to be included. The configuration ofany carbon-carbon double bond appearing herein is selected forconvenience only and is not intended to limit a particular configurationunless the text so states.

The terms “substituent” and “substituent group,” as used herein, aremeant to include groups that are typically added to a parent compoundsor to further substituted substituent groups to enhance one or moredesired properties or provide other desired effects. Substituent groupscan be protected or unprotected and can be added to one available siteor many available sites on a parent compound. As an example if a benzeneis substituted with a substituted alky it will not have any overlap witha benzene that is substituted with substituted hydroxyl. In such anexample the alkyl portion of the substituted alkyl is covalently linkedby one of its carbon atoms to one of the benzene carbon atoms. If thealky is C₁ and it is substituted with a hydroxyl substituent group(substituted alkyl) then the resultant compound is benzyl alcohol(C₆H₅CH₂OH). If the benzene were substituted with a substituted hydroxylgroup and the hydroxyl was substituted with a C₁ alkyl group then theresultant compound would be anisole (C₆H₅OCH₃).

Substituent groups amenable herein include without limitation, halogen,hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R_(aa)), carboxyl(—C(O)O—R_(aa)), aliphatic groups, alicyclic groups, alkoxy, substitutedoxy (—O—R_(aa)), aryl, aralkyl, heterocyclic radical, heteroaryl,heteroarylalkyl, amino (—N(R_(bb))(R_(cc))), imino(═NR_(bb)), amido(—C(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro(—NO₂), cyano (—CN), carbamido (—OC(O)N(R_(bb))(R_(cc)) or—N(R_(bb))C(O)OR_(aa)), ureido (—N(R_(bb))C(O)—N(R_(bb))(R_(cc))),thioureido (—N(R_(bb))C(S)N(R_(bb))(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S(O)₂R_(bb)). Whereineach R_(aa), R_(bb) and R_(cc) is, independently, H, an optionallylinked chemical functional group or a further substituent group with apreferred list including without limitation, H, alkyl, alkenyl, alkynyl,aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,heterocyclic and heteroarylalkyl. Selected substituents within thecompounds described herein are present to a recursive degree.

In this context, “recursive substituent” means that a substituent mayrecite another instance of itself. Because of the recursive nature ofsuch substituents, theoretically, a large number may be present in anygiven claim. One of ordinary skill in the art of medicinal chemistry andorganic chemistry understands that the total number of such substituentsis reasonably limited by the desired properties of the compoundintended. Such properties include, by way of example and not limitation,physical properties such as molecular weight, solubility or logP,application properties such as activity against the intended target andpractical properties such as ease of synthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in a claim of the invention, the total numberwill be determined as set forth above.

The terms “stable compound” and “stable structure” as used herein aremeant to indicate a compound that is sufficiently robust to surviveisolation to a useful degree of purity from a reaction mixture, andformulation into an efficacious therapeutic agent. Only stable compoundsare contemplated herein.

As used herein the term “nucleobase” generally refers to the nucleobaseof a nucleoside or modified nucleoside. The term “heterocyclic basemoiety” is broader than the term nucleobase in that it includes anyheterocyclic base that can be attached to a sugar to prepare anucleoside or modified nucleoside. Such heterocyclic base moietiesinclude but are not limited to naturally occurring nucleobases (adenine,guanine, thymine, cytosine and uracil) and protected forms of unmodifiednucleobases (4-N-benzoylcytosine, 6-N-benzoyladenine and2-N-isobutyrylguanine) as well as modified (5-methyl cytosine) ornon-naturally occurring heterocyclic base moieties and syntheticmimetics thereof (such as for example phenoxazines).

In one embodiment, a heterocyclic base moiety is any heterocyclic systemthat contains one or more atoms or groups of atoms capable of hydrogenbonding to a heterocyclic base of a nucleic acid. In certainembodiments, nucleobase refers to purines, modified purines, pyrimidinesand modified pyrimidines. In certain embodiments, nucleobase refers tounmodified or naturally occurring nucleobases which include, but are notlimited to, the purine bases adenine (A) and guanine (G), and thepyrimidine bases thymine (T), cytosine (C) and uracil (U) and analogsthereof such as 5-methyl cytosine. The terms nucleobase and heterocyclicbase moiety also include optional protection for any reactive functionalgroups such as 4-N-benzoylcytosine, 4-N-benzoyl-5-methylcytosine,6-N-benzoyladenine or 2-N-isobutyrylguanine

In certain embodiments, heterocyclic base moieties include withoutlimitation modified nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein.

In certain embodiments, heterocyclic base moieties include withoutlimitation tricyclic pyrimidines such as 1,3-diazaphenoxazine-2-one,1,3-diazaphenothiazine-2-one and9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Heterocyclicbase moieties also include those in which the purine or pyrimidine baseis replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further heterocyclicbase moieties include without limitation those known to the art skilled(see for example: U.S. Pat. No. 3,687,808; Swayze et al., The MedicinalChemistry of Oligonucleotides in Antisense a Drug Technology, Chapter 6,pages 143-182, Crooke, S. T., ed., 2008); The Concise Encyclopedia OfPolymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley &Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, InternationalEdition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Researchand Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993,273-302). Modified polycyclic heterocyclic compounds useful asheterocyclic base moieties are disclosed in the above noted U.S. Pat.No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,681,941;5,750,692; 5,763,588; 5,830,653; 6,005,096; and U.S. Patent ApplicationPublication 20030158403, each of which is incorporated herein byreference in its entirety.

As used herein the term “sugar moiety” refers to naturally occurringsugars having a furanose ring system (ribose and 2′-deoxyribose),synthetic and/or non-naturally occurring sugars having a modifiedfuranose ring system and sugar surrogates wherein the furanose ring hasbeen replaced with a mono or polycyclic ring system such as for examplea morpholino or hexitol ring system or a non-cyclic sugar surrogate suchas that used in peptide nucleic acids. The sugar moiety of a monomersubunit provides the reactive groups that enable the linking of adjacentmonomer subunits into an oligomeric compound. Illustrative examples ofsugar moieties useful in the preparation of oligomeric compounds includewithout limitation, β-D-ribose, β-D-2′-deoxyribose, substituted sugars(such as 2′, 5′ and bis substituted sugars), 4′-S-sugars (such as4′-S-ribose, 4′-S-2′-deoxyribose and 4′-S-2′-substituted ribose whereinthe ring oxygen atom has been replaced with a sulfur atom), bicyclicmodified sugars (such as the 2′-O—CH(CH₃)-4′,2′-O—CH₂-4′ or2′-O—(CH₂)₂-4′ bridged ribose derived bicyclic sugars) and sugarsurrogates (such as for example when the ribose ring has been replacedwith a morpholino, a hexitol ring system or an open non-cyclic system).

As used herein the term “sugar surrogate” refers to replacement of thenucleoside furanose ring with a non-furanose (or 4′-substitutedfuranose) group with another structure such as another ring system oropen system. Such structures can be as simple as a six membered ring asopposed to the five membered furanose ring or can be more complicatedsuch as a bicyclic or tricyclic ring system or a non-ring system used inpeptide nucleic acid. In certain embodiments, sugar surrogates includewithout limitation sugar surrogate groups such as morpholinos,cyclohexenyls and cyclohexitols. In general the heterocyclic base ismaintained even when the sugar moiety is a sugar surrogate so that theresulting monomer subunit will be able to hybridize.

As used herein the term “sugar substituent group” refers to a group thatis covalently attached to a sugar moiety. In certain embodiments,examples of sugar substituent groups include without limitation halogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, amino, substitutedamino, thio, substituted thio and azido. In certain embodiments thealkyl and alkoxy groups are C₁ to C₆. In certain embodiments, thealkenyl and alkynyl groups are C₂ to C₆. In certain embodiments,examples of sugar substituent groups include without limitation 2′-F,2′-allyl, 2′-amino, 2′-azido, 2′-thio, 2′-O-allyl, 2′-OCF₃, 2′-O—C₁-C₁₀alkyl, 2′-OCH₃, 2′—O(CH₂)_(n)CH₃, 2′-OCH₂CH₃, 2′-O—(CH₂)₂CH₃,2′-O—(CH₂)₂—O—CH₃ (MOE), 2′—O[(CH₂)_(n)O]_(m)CH₃, 2′-O(CH₂)₂SCH₃,2′-O—(CH₂)₃—N(R_(p))(R_(q)), 2′—O(CH₂)_(n)NH₂,2′-O—(CH₂)₂—O—N(R_(p))(R_(q)), O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂,2′—O(CH₂)_(n)ONH₂, 2′-O—(CH₂)₂—O—(CH₂)₂—N(R_(p))(R_(q)),2′—O—CH₂C(═O)—N(R_(p))(R_(q)), 2′-OCH₂C(═O)N(H)CH₃,2′-O—CH₂C(═O)—N(H)—(CH₂)₂—N(R_(p))(R_(q)) and2′-O—CH₂—N(H)—C(═NR_(r))[N(R_(p))(R_(q))], wherein each R_(p), R_(q) andR_(r) is, independently, H, substituted or unsubstituted C₁-C₁₀ alkyl ora protecting group and where n and m are from 1 to about 10.

In certain embodiments, examples of sugar substituent groups includewithout limitation 2′-F, 2′-allyl, 2′-amino, 2′-azido, 2′-thio,2′-O-allyl, 2′-OCF₃, 2′-O—C₁-C₁₀ alkyl, 2′-O—CH₃, OCF₃, 2′—O—CH₂CH₃,2′-O—(CH₂)₂CH₃, 2′-O—(CH₂)₂—O—CH₃ (MOE), 2′-O(CH₂)₂SCH₃,2′-O—CH₂—CH═CH₂, 2′—O—(CH₂)₃—N(R_(m))(R_(n)),2′-O—(CH₂)₂—O—N(R_(m))(R_(n)), 2′-O—(CH₂)₂—O—(CH₂)₂—N(R_(m))(R_(n)),2′-O—CH₂C(═O)—N(R_(m))(R_(n)), 2′-O—CH₂C(═O)—N(H)—(CH₂)₂—N(R_(m))(R_(n))and 2′-O—CH₂—N(H)—C(═NR_(m))[N(R_(m))(R_(n))] wherein each R_(m) andR_(n) is, independently, H, substituted or unsubstituted C₁-C₁₀ alkyl ora protecting group. In certain embodiments, examples of 2,-sugarsubstituent groups include without limitation fluoro, —O—CH₃, —O—CH₂CH₃,—O—(CH₂)₂CH₃, —O—(CH₂)₂—O—CH₃, —O—CH₂—CH═CH₂, —O—(CH₂)₃—N(R₁)(R₂),O—(CH₂)₂—O—N(R₁)(R₂), —O—(CH₂)₂—O—(CH₂)₂—N(R₁)(R₂),—O—CH₂C(═O)—N(R₁)(R₂), —O—CH₂C(═O)—N(H)—(CH₂)₂—N(R₁)(R₂) and—O—CH₂—N(H)—C(═NR₁)[N(R₁)(R₂)] wherein R₁ and R₂ are each independently,H or C₁-C₂ alkyl. In certain embodiments, examples of sugar substituentgroups include without limitation fluoro, —O—CH₃, —O—(CH₂)₂—O—CH₃,—O—CH₂C(═O)—N(H)(CH₃), —O—CH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ and—O—CH₂—N(H)—C(═NCH₃)[N(CH₃)₂]. In certain embodiments, examples of sugarsubstituent groups include without limitation fluoro, —O—CH₃,—O—(CH₂)₂—O—CH₃, —O—CH₂C(═O)—N(H)(CH₃) and—O—CH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂. Further examples of modified sugarmoieties include without limitation bicyclic sugars (e.g. bicyclicnucleic acids or bicyclic nucleosides discussed below).

In certain embodiments, sugar substituent groups include withoutlimitation one or two 5′-sugar substituent groups independently selectedfrom C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substitutedC₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl and halogen. Incertain embodiments, examples of sugar substituent groups includewithout limitation one or two 5′-sugar substituent groups independentlyselected from vinyl, 5′-methyl, 5′-(S)-methyl and 5′-(R)-methyl. Incertain embodiments, examples of sugar substituent groups includewithout limitation one 5′-sugar substituent group selected from vinyl,5′-(S)-methyl and 5′-(R)-methyl.

In certain embodiments, examples of sugar substituent groups includewithout limitation substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving pharmacokineticproperties, or a group for improving the pharmacodynamic properties ofan oligomeric compound, and other substituents having similarproperties. In certain embodiments, oligomeric compounds includemodified nucleosides comprising 2′-MOE substituent groups (Baker et al.,J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution hasbeen described as having improved binding affinity compared tounmodified nucleosides and to other modified nucleosides, such as2′-O-methyl, 2′-O-propyl, and 2′-O-aminopropyl. Oligonucleotides havingthe 2′-MOE substituent also have been shown to be antisense inhibitorsof gene expression with promising features for in vivo use (Martin, P.,Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50,168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; andAltmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

Sugar moieties can be substituted with more than one sugar substituentgroup including without limitation 2′-F-5′-methyl substitutednucleosides (see PCT International Application WO 2008/101157, publishedon Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides).Other combinations are also possible, including without limitation,replacement of the ribosyl ring oxygen atom with S and furthersubstitution at the 2′-position (see published U.S. Patent ApplicationUS2005-0130923, published on Jun. 16, 2005) and 5′-substitution of abicyclic nucleoside (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup).

As used herein the term “monomer subunit” is meant to include all mannerof monomers that are amenable to oligomer synthesis. In general amonomer subunit includes at least a sugar moiety having at least tworeactive sites that can form linkages to further monomer subunits.Essentially all monomer subunits include a heterocyclic base moiety thatis hybridizable to a complementary site on a nucleic acid target.Reactive sites on monomer subunits located on the termini of anoligomeric compound can be protected or unprotected (generally OH) orcan form an attachment to a terminal group (conjugate or other group).Monomer subunits include, without limitation, nucleosides and modifiednucleosides. In certain embodiments, monomer subunits includenucleosides such as 13-D-ribonucleosides and β-D-2′-deoxyribnucleosidesand modified nucleosides including but not limited to substitutednucleosides (such as 2′, 5′ and bis substituted nucleosides),4′-S-modified nucleosides (such as 4′-S-ribonucleosides,4′-S-2′-deoxyribonucleosides and 4′-S-2′-substituted ribonucleosides),bicyclic modified nucleosides (such as bicyclic nucleosides wherein thesugar moiety has a 2′-O—CHR_(a)-4′ bridging group, wherein R_(a) is H,alkyl or substituted alkyl), other modified nucleosides and nucleosideshaving sugar surrogates. As used herein, the term “nucleoside” refers toa nucleobase-sugar combination. The two most common classes of suchnucleobases are purines and pyrimidines. The term nucleoside includesβ-D-ribonucleosides and β-D-2′-deoxyribonucleosides.

As used herein, the term “nucleotide” refers to a nucleoside furthercomprising a modified or unmodified phosphate internucleoside linkinggroup or a non-phosphate internucleoside linking group. For nucleotidesthat include a pentofuranosyl sugar, the internucleoside linking groupcan be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar.The phosphate and or a non-phosphate internucleoside linking groups areroutinely used to covalently link adjacent nucleosides to one another toform a linear polymeric compound.

As used herein the term “modified nucleoside” refers to a nucleosidecomprising a modified heterocyclic base and or a sugar moiety other thanribose and 2′-deoxyribose. In certain embodiments, a modified nucleosidecomprises a modified heterocyclic base moiety. In certain embodiments, amodified nucleoside comprises a sugar moiety other than ribose and2′-deoxyribose. In certain embodiments, a modified nucleoside comprisesa modified heterocyclic base moiety and a sugar moiety other than riboseand 2′-deoxyribose. The term “modified nucleoside” is intended toinclude all manner of modified nucleosides that can be incorporated intoan oligomeric compound using standard oligomer synthesis protocols.Modified nucleosides include abasic nucleosides but in general aheterocyclic base moiety is included for hybridization to acomplementary nucleic acid target.

In certain embodiments, modified nucleosides include a furanose ringsystem or a modified furanose ring system. Modified furanose ringsystems include 4′-S analogs, one or more substitutions at any positionsuch as for example the 2′, 3′, 4′ and 5′ positions and addition ofbridges for form additional rings such as a 2′-O—CH(CH₃)-4′ bridge. Suchmodified nucleosides include without limitation, substituted nucleosides(such as 2′, 5′, and/or 4′ substituted nucleosides) 4′-S-modifiednucleosides, (such as 4′-S-ribonucleosides, 4′-S-2′-deoxyribonucleosidesand 4′-5-2′-substituted ribonucleosides), bicyclic modified nucleosides(such as 2′-O—CH(CH₃)-4′,2′-O—CH₂-4′ or 2′-O—(CH₂)₂-4′ bridged furanoseanalogs) and base modified nucleosides. The sugar can be modified withmore than one of these modifications listed such as for example abicyclic modified nucleoside further including a 5′-substitution or a 5′or 4′ substituted nucleoside further including a 2′ substituent. Theterm modified nucleoside also includes combinations of thesemodifications such as base and sugar modified nucleosides. Thesemodifications are meant to be illustrative and not exhaustive as othermodifications are known in the art and are also envisioned as possiblemodifications for the modified nucleosides described herein.

In certain embodiments, modified nucleosides comprise a sugar surrogatewherein the furanose ring has been replaced with a mono or polycyclicring system or a non-cyclic sugar surrogate such as that used in peptidenucleic acids. Illustrative examples of sugar moieties for such modifiednucleosides includes without limitation morpholino, hexitol,cyclohexenyl, 2.2.2 and 3.2.1 cyclohexose and open non-cyclic groups.

In certain embodiments, modified nucleosides comprise a non-naturallyoccurring sugar moiety and a modified heterocyclic base moiety. Suchmodified nucleosides include without limitation modified nucleosideswherein the heterocyclic base moiety is replaced with a phenoxazinemoiety (for example the 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-onegroup, also referred to as a G-clamp which forms four hydrogen bondswhen hybridized with a guanosine base) and further replacement of thesugar moiety with a sugar surrogate group such as for example amorpholino, a cyclohexenyl or a bicyclo[3.1.0]hexyl.

As used herein the term “bicyclic nucleoside” refers to a nucleosidecomprising at least a bicyclic sugar moiety. Examples of bicyclicnucleosides include without limitation nucleosides having a furanosylsugar that comprises a bridge between two of the non-geminal carbonsatoms. In certain embodiments, bicyclic nucleosides have a bridgebetween the 4′ and 2′ carbon atoms. Examples of such 4′ to 2′ bridgedbicyclic nucleosides, include but are not limited to one of formulae:4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′and 4′-C—H(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No.7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogsthereof see published International Application WO/2009/006478,published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof seepublished International Application WO2008/150729, published Dec. 11,2008); 4′-CH₂—O—N(CH₃)-2′ (see U.S. Pat. No. 7,96,345, issued on Apr.13, 2010,); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or aprotecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008);4′-CH₂—C(H)(CH₃)-2′ (see Chattopadhyaya, et al., J. Org. Chem., 2009,74, 118-134); and 4′-CH₂—CH₂-2′ and 4′-CH₂—C—(═CH₂)-2′ (and analogsthereof see published International Application WO 2008/154401,published on Dec. 8, 2008). Further bicyclic nucleosides have beenreported in published literature (see for example: Srivastava et al., J.Am. Chem. Soc., 2007, 129(26) 8362-8379; Frieden et al., Nucleic AcidsResearch, 2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens.Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orumet al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wahlestedt et al.,Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Singh et al., Chem.Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222;Singh et al., J. Org. Chem., 1998, 63, 10035-10039; U.S. Pat. Nos.7,741,457; 7,696,345; 7,547,684,;7,399,845; 7,053,207; 7,034,133;6,794,499; 6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S. PatentPublication Nos.: US2008-0039618; U.S. Patent Applications, Ser. Nos.61/099,844; 61/097,787; 61/086,231; 61/056,564; 61/026,998; 61/026,995;60/989,574; International applications WO2009/006478; WO2008/154401;WO2008/150729; WO 2007/134181; WO 2005/021570; WO 2004/106356; WO94/14226). Each of the foregoing bicyclic nucleosides can be preparedhaving one or more stereochemical sugar configurations including forexample α-L-ribofuranose and β-D-ribofuranose (see PCT internationalapplication PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic nucleosides comprise a bridge betweenthe 4′ and the 2′ carbon atoms of the pentofuranosyl sugar moietyincluding without limitation, bridges comprising 1 or from 1 to 4 linkedgroups (generally forming a 4 to 6 membered ring with the parent sugarmoiety) independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein: x is 0, 1, or 2; nis 1, 2, 3, or 4; each R_(a) and R_(b) is, independently, H, aprotecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycleradical, substituted heterocycle radical, heteroaryl, substitutedheteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclicradical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl(C(═O)—H), substitutedacyl, CN, sulfonyl(S(═O)₂-J₁), or sulfoxyl(S(═O)-J₁); and each J₁ and J₂is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H),substituted acyl, a heterocycle radical, a substituted heterocycleradical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or aprotecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′,4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′-wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a4′-(CH₂)—O-2′ bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy(4′-CH₂—O-2′) BNA's have beenincorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include those having a 4′to 2′ bridge wherein such bridges include without limitation,α-L-4′-(CH₂)—O-2′, β-D-4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′,4′-CH₂—O—N(R)-2′,4′-CH₂—N(R)—O-2′, 4′-CH(CH₃)—O-2′, 4′-CH₂—S-2′,4′-CH₂—N(R)-2′, 4′-CH₂—CH(CH₃)-2′, and 4′-(CH₂)₃-2′, wherein R is H, aprotecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thiol.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl(C(═O)—).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl.

The synthesis and preparation of adenine, cytosine, guanine,5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a4′-CH₂—O-2′ bridge, along with their oligomerization, and nucleic acidrecognition properties have been described (Koshkin et al., Tetrahedron,1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has alsobeen described in WO 98/39352 and WO 99/14226.

Analogs of various bicyclic nucleosides that have 4′ to 2′ bridginggroups such as 4′-CH₂—O-2′ and 4′-CH₂—S-2′, have also been prepared(Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).Preparation of oligodeoxyribonucleotide duplexes comprising bicyclicnucleosides for use as substrates for nucleic acid polymerases has alsobeen described (Wengel et al., WO 99/14226). Furthermore, synthesis of2′-amino-BNA, a novel conformationally restricted high-affinityoligonucleotide analog has been described in the art (Singh et al., J.Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and2′-methylamino-BNA's have been prepared and the thermal stability oftheir duplexes with complementary RNA and DNA strands has beenpreviously reported.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Frier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-methyleneoxy(4′-CH₂—O-2′) BNA, (B)β-D-methyleneoxy(4′-CH₂—O-2) BNA (C) ethyleneoxy(4′-(CH₂)₂—O-2′) BNA (D)aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino(4′-CH₂—N(R)—O-2′) BNA, (F)methyl(methyleneoxy) (4′-CH(CH₃)—O-2) BNA (also referred to asconstrained ethyl or cEt), (G) methylene-thio(4′-CH₂—S-2′) BNA, (H)methylene-amino(4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic(4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA,and (K) vinyl BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protectinggroup, C₁-C₆ alkyl or C₁-C₆ alkoxy.

In certain embodiments, modified nucleosides include nucleosides havingsugar surrogate groups that include without limitation, replacement ofthe ribosyl ring with a sugar surrogate such as a tetrahydropyranyl ringsystem (also referred to as hexitol) as illustrated below:

In certain embodiments, sugar surrogates are selected having theformula:

wherein:

Bx is a heterocyclic base moiety;

one of T₃ and T₄ is an internucleoside linking group attaching thetetrahydropyran nucleoside analog to the remainder of one of the 5′ or3′ end of the oligomeric compound and the other of T₃ and T₄ ishydroxyl, a protected hydroxyl, a 5′ or 3′ terminal group or aninternucleoside linking group attaching the tetrahydropyran nucleosideanalog to the remainder of the other of the 5′ or 3′ end of theoligomeric compound;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and

one of R₁ and R₂ is hydrogen and the other is selected from halogen,substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁,J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isother than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄,q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Such sugar surrogates can be referred to as a “modified tetrahydropyrannucleoside” or “modified THP nucleoside”. Modified THP nucleosidesinclude, but are not limited to, what is referred to in the art ashexitol nucleic acid (HNA), altritol nucleic acid (ANA), and mannitolnucleic acid (MNA) (see Leumann, C. J., Bioorg. & Med. Chem., 2002, 10,841-854).

In certain embodiments, oligomeric compounds comprise one or moremodified cyclohexenyl nucleosides, which is a nucleoside having asix-membered cyclohexenyl in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified cyclohexenyl nucleosidesinclude, but are not limited to those described in the art (see forexample commonly owned, published PCT Application WO 2010/036696,published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008,130(6), 1979-1984; Horváth et al., Tetrahedron Letters, 2007, 48,3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30),9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005,24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005,33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F:Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenylnucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

one of T₃ and T₄ is an internucleoside linking group attaching thecyclohexenyl nucleoside analog to the remainder of one of the 5′ or 3′end of the oligomeric compound and the other of T₃ and T₄ is hydroxyl, aprotected hydroxyl, a 5′ or 3′ terminal group or an internucleosidelinking group attaching the cyclohexenyl nucleoside analog to theremainder of the other of the 5′ or 3′ end of the oligomeric compound;and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugarsubstituent group.

Many other monocyclic, bicyclic and tricyclic ring systems are known inthe art and are suitable as sugar surrogates that can be used to modifynucleosides for incorporation into oligomeric compounds as providedherein (see for example review article: Leumann, Christian J. Bioorg. &Med. Chem., 2002, 10, 841-854). Such ring systems can undergo variousadditional substitutions to further enhance their activity.

Some representative U.S. patents that teach the preparation of suchmodified sugars include without limitation, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920;5,792,847 and 6,600,032 and International Application PCT/US2005/019219,filed Jun. 2, 2005 and published as WO 2005/121371 on Dec. 22, 2005certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The 2′-thio bicyclic nucleosides provided herein can be prepared by anyof the applicable techniques of organic synthesis, as, for example,illustrated in the examples below. Many such techniques are well knownin the art. However, many of the known techniques are elaborated inCompendium of Organic Synthetic Methods, John Wiley & Sons, New York:Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T.Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and LeroyWade, 1977; Vol. 4, Leroy G. Wade Jr., 1980; Vol. 5, Leroy G. Wade Jr.,1984; and Vol. 6, Michael B. Smith; as well as March, J., AdvancedOrganic Chemistry, 3rd Edition, John Wiley & Sons, New York, 1985;Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency inModern Organic Chemistry, in 9 Volumes, Barry M. Trost, Editor-in-Chief,Pergamon Press, New York, 1993; Advanced Organic Chemistry, Part B:Reactions and Synthesis, 4th Edition; Carey and Sundberg, KluwerAcademic/Plenum Publishers, New York, 2001; Advanced Organic Chemistry,Reactions, Mechanisms, and Structure, 2nd Edition, March, McGraw Hill,1977; Greene, T. W., and Wutz, P. G. M., Protecting Groups in OrganicSynthesis, 4th Edition, John Wiley & Sons, New York, 1991; and Larock,R. C., Comprehensive Organic Transformations, 2nd Edition, John Wiley &Sons, New York, 1999.

As used herein the term “reactive phosphorus” is meant to include groupsthat are covalently linked to a monomer subunit that can be furtherattached to an oligomeric compound that are useful for forminginternucleoside linkages including for example phosphodiester andphosphorothioate internucleoside linkages. Such reactive phosphorusgroups are known in the art and contain phosphorus atoms in P^(III) orP^(V) valence state including, but not limited to, phosphoramidite,H-phosphonate, phosphate triesters and phosphorus containing chiralauxiliaries. In certain embodiments, reactive phosphorus groups areselected from diisopropylcyanoethoxy phosphoramidite(—O*—P[N[(CH(CH₃)₂]₂]O(CH₂)₂CN) and H-phosphonate (—O*—P(═O)(H)OH),wherein the O* is normally attached to the 3′-position of the Markushgroup of Formula II or IIa. A preferred synthetic solid phase synthesisutilizes phosphoramidites (P^(III) chemistry) as reactive phosphites.The intermediate phosphite compounds are subsequently oxidized to thephosphate or thiophosphate (P^(V) chemistry) using known methods toyield, phosphodiester or phosphorothioate internucleoside linkages.Chiral auxiliaries are known in the art (see for example: Wang et al.,Tetrahedron Letters, 1997, 38(5), 705-708; Jin et al., J. Org. Chem.,1997, 63, 3647-3654; Wang et al., Tetrahedron Letters, 1997, 38(22),3797-3800; and U.S. Pat. No. 6,867,294, issued Mar. 15, 2005).Additional reactive phosphates and phosphites are disclosed inTetrahedron Report Number 309 (Beaucage and Iyer, Tetrahedron, 1992, 48,2223-2311).

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

The term “oligonucleoside” refers to a sequence of nucleosides that arejoined by internucleoside linkages that do not have phosphorus atoms.Internucleoside linkages of this type include short chain alkyl,cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one ormore short chain heteroatomic and one or more short chain heterocyclic.These internucleoside linkages include without limitation, siloxane,sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl,methylene formacetyl, thioformacetyl, alkeneyl, sulfamate,methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide andothers having mixed N, O, S and CH₂ component parts.

As used herein, the term “oligomeric compound” refers to a contiguoussequence of linked monomer subunits. Each linked monomer subunitnormally includes a heterocyclic base moiety but monomer subunits alsoincludes those without a heterocyclic base moiety such as abasic monomersubunits. At least some and generally most if not essentially all of theheterocyclic bases in an oligomeric compound are capable of hybridizingto a nucleic acid molecule, normally a preselected RNA target. The term“oligomeric compound” therefore includes oligonucleotides,oligonucleotide analogs and oligonucleosides. It also includes polymershaving one or a plurality of nucleosides having sugar surrogate groups.

In certain embodiments, oligomeric compounds comprise a plurality ofmonomer subunits independently selected from naturally occurringnucleosides, non-naturally occurring nucleosides, modified nucleosidesand nucleosides having sugar surrogate groups. In certain embodiments,oligomeric compounds are single stranded. In certain embodiments,oligomeric compounds are double stranded comprising a double-strandedduplex. In certain embodiments, oligomeric compounds comprise one ormore conjugate groups and/or terminal groups.

When preparing oligomeric compounds having specific motifs as disclosedherein it can be advantageous to mix non-naturally occurring monomersubunits such as the 2′-thio bicyclic nucleosides as provided hereinwith other non-naturally occurring monomer subunits, naturally occurringmonomer subunits (nucleosides) or mixtures thereof. In certainembodiments, oligomeric compounds are provided herein comprising acontiguous sequence of linked monomer subunits wherein at least onemonomer subunit is a 2′-thio bicyclic nucleoside as provided herein. Incertain embodiments, oligomeric compounds are provided comprising aplurality of 2′-thio bicyclic nucleosides as provided herein.

Oligomeric compounds are routinely prepared linearly but can also bejoined or otherwise prepared to be circular and/or can be prepared toinclude branching. Oligomeric compounds can form double strandedconstructs such as for example two strands hybridized to form a doublestranded composition. Double stranded compositions can be linked orseparate and can include various other groups such as conjugates and/oroverhangs on the ends.

As used herein, “antisense compound” refers to an oligomeric compound,at least a portion of which is at least partially complementary to atarget nucleic acid to which it hybridizes. In certain embodiments, anantisense compound modulates (increases or decreases) expression oramount of a target nucleic acid. In certain embodiments, an antisensecompound alters splicing of a target pre-mRNA resulting in a differentsplice variant. In certain embodiments, an antisense compound modulatesexpression of one or more different target proteins. Antisensemechanisms contemplated herein include, but are not limited to an RNaseH mechanism, RNAi mechanisms, splicing modulation, translational arrest,altering RNA processing, inhibiting microRNA function, or mimickingmicroRNA function.

As used herein, “antisense activity” refers to any detectable and/ormeasurable activity attributable to the hybridization of an antisensecompound to its target nucleic acid. In certain embodiments, suchactivity may be an increase or decrease in an amount of a nucleic acidor protein. In certain embodiments, such activity may be a change in theratio of splice variants of a nucleic acid or protein. Detection and/ormeasuring of antisense activity may be direct or indirect. For example,in certain embodiments, antisense activity is assessed by detectingand/or measuring the amount of target protein or the relative amounts ofsplice variants of a target protein. In certain embodiments, antisenseactivity is assessed by detecting and/or measuring the amount of targetnucleic acids and/or cleaved target nucleic acids and/or alternativelyspliced target nucleic acids. In certain embodiments, antisense activityis assessed by observing a phenotypic change in a cell or animal.

As used herein the term “internucleoside linkage” or “internucleosidelinking group” is meant to include all manner of internucleoside linkinggroups known in the art including but not limited to, phosphoruscontaining internucleoside linking groups such as phosphodiester andphosphorothioate, and non-phosphorus containing internucleoside linkinggroups such as formacetyl and methyleneimino. Internucleoside linkagesalso includes neutral non-ionic internucleoside linkages such as amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′) andmethylphosphonate wherein a phosphorus atom is not always present.

In certain embodiments, oligomeric compounds as provided herein can beprepared having one or more internucleoside linkages containing modifiede.g. non-naturally occurring internucleoside linkages. The two mainclasses of internucleoside linkages are defined by the presence orabsence of a phosphorus atom. Modified internucleoside linkages having aphosphorus atom include without limitation, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity can comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus containing linkages include without limitation, U.S. Pat.Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;5,194,599; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,527,899; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,565,555; 5,571,799; 5,587,361; 5,625,050; 5,672,697 and 5,721,218,certain of which are commonly owned with this application, and each ofwhich is herein incorporated by reference.

In certain embodiments, oligomeric compounds as provided herein can beprepared having one or more non-phosphorus containing internucleosidelinkages. Such oligomeric compounds include without limitation, thosethat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages. These include those having siloxane backbones;sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; riboacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include without limitation, U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,677,439;5,646,269 and 5,792,608, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

As used herein “neutral internucleoside linkage” is intended to includeinternucleoside linkages that are non-ionic. Neutral internucleosidelinkages include without limitation, phosphotriesters,methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

In certain embodiments, oligomeric compounds as provided herein can beprepared having one or more optionally protected phosphorus containinginternucleoside linkages. Representative protecting groups forphosphorus containing internucleoside linkages such as phosphodiesterand phosphorothioate linkages include β-cyanoethyl, diphenylsilylethyl,δ-cyanobutenyl, cyano p-xylyl (CPX), N-methyl-N-trifluoroacetyl ethyl(META), acetoxy phenoxy ethyl (APE) and butene-4-yl groups. See forexample U.S. Pat. Nos. 4,725,677 and Re. 34,069 (β-cyanoethyl); Beaucageet al., Tetrahedron, 1993, 49(10), 1925-1963; Beaucage et al.,Tetrahedron, 1993, 49(46), 10441-10488; Beaucage et al., Tetrahedron,1992, 48(12), 2223-2311.

As used herein the terms “linking groups” and “bifunctional linkingmoieties” are meant to include groups known in the art that are usefulfor attachment of chemical functional groups, conjugate groups, reportergroups and other groups to selective sites in a parent compound such asfor example an oligomeric compound. In general, a bifunctional linkingmoiety comprises a hydrocarbyl moiety having two functional groups. Oneof the functional groups is selected to bind to a parent molecule orcompound of interest and the other is selected to bind to essentiallyany selected group such as a chemical functional group or a conjugategroup. In some embodiments, the linker comprises a chain structure or apolymer of repeating units such as ethylene glycols or amino acid units.Examples of functional groups that are routinely used in bifunctionallinking moieties include without limitation, electrophiles for reactingwith nucleophilic groups and nucleophiles for reacting withelectrophilic groups. In some embodiments, bifunctional linking moietiesinclude amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g.,double or triple bonds), and the like. Some nonlimiting examples ofbifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid(ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groupsinclude without limitation, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, the oligomeric compounds as provided herein canbe modified by covalent attachment of one or more conjugate groups. Ingeneral, conjugate groups modify one or more properties of theoligomeric compounds they are attached to. Such oligonucleotideproperties include without limitation, pharmacodynamics,pharmacokinetics, binding, absorption, cellular distribution, cellularuptake, charge and clearance. Conjugate groups are routinely used in thechemical arts and are linked directly or via an optional linking moietyor linking group to a parent compound such as an oligomeric compound. Apreferred list of conjugate groups includes without limitation,intercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, thioethers, polyethers, cholesterols, thiocholesterols, cholicacid moieties, folate, lipids, phospholipids, biotin, phenazine,phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,rhodamines, coumarins and dyes.

In certain embodiments, the oligomeric compounds as provided herein canbe modified by covalent attachment of one or more terminal groups to the5′ or 3′-terminal groups. A terminal group can also be attached at anyother position at one of the terminal ends of the oligomeric compound.As used herein the terms “5′-terminal group”, “3′-terminal group”,“terminal group” and combinations thereof are meant to include usefulgroups known to the art skilled that can be placed on one or both of theterminal ends, including but not limited to the 5′ and 3′-ends of anoligomeric compound respectively, for various purposes such as enablingthe tracking of the oligomeric compound (a fluorescent label or otherreporter group), improving the pharmacokinetics or pharmacodynamics ofthe oligomeric compound (such as for example: uptake and/or delivery) orenhancing one or more other desirable properties of the oligomericcompound (a group for improving nuclease stability or binding affinity).In certain embodiments, 5′ and 3′-terminal groups include withoutlimitation, modified or unmodified nucleosides; two or more linkednucleosides that are independently, modified or unmodified; conjugategroups; capping groups; phosphate moieties; and protecting groups.

As used herein the term “phosphate moiety” refers to a terminalphosphate group that includes phosphates as well as modified phosphates.The phosphate moiety can be located at either terminus but is preferredat the 5′-terminal nucleoside. In one aspect, the terminal phosphate isunmodified having the formula —O—P(═O)(OH)OH. In another aspect, theterminal phosphate is modified such that one or more of the O and OHgroups are replaced with H, O, S, N(R) or alkyl where R is H, an aminoprotecting group or unsubstituted or substituted alkyl. In certainembodiments, the 5′ and or 3′ terminal group can comprise from 1 to 3phosphate moieties that are each, independently, unmodified (di ortri-phosphates) or modified.

As used herein, the term “phosphorus moiety” refers to a group havingthe formula:

wherein:

R_(x) and R_(y) are each, independently, hydroxyl, protected hydroxylgroup, thiol, protected thiol group, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, a protected amino orsubstituted amino; and

R_(z) is O or S.

As a monomer such as a phosphoramidite or H-phosphonate the protectedphosphorus moiety is preferred to maintain stability during oligomersynthesis. After incorporation into an oligomeric compound thephosphorus moiety can include deprotected groups.

Phosphorus moieties included herein can be attached to a monomer, whichcan be used in the preparation of oligomeric compounds, wherein themonomer may be attached using O, S, NR_(d) or CR_(e)R_(f), wherein R_(d)includes without limitation H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl,C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or substituted acyl,and R_(e) and R_(f) each, independently, include without limitation H,halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy orsubstituted C₁-C₆ alkoxy. Such linked phosphorus moieties includewithout limitation, phosphates, modified phosphates, thiophosphates,modified thiophosphates, phosphonates, modified phosphonates,phosphoramidates and modified phosphoramidates.

RNA duplexes exist in what has been termed “A Form” geometry while DNAduplexes exist in “B Form” geometry. In general, RNA:RNA duplexes aremore stable, or have higher melting temperatures (T_(m)) than DNA:DNAduplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984,Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34,10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). Theincreased stability of RNA has been attributed to several structuralfeatures, most notably the improved base stacking interactions thatresult from an A-form geometry (Searle et al., Nucleic Acids Res., 1993,21, 2051-2056). The presence of the 2′ hydroxyl in RNA biases the sugartoward a C3′ endo pucker, i.e., also designated as Northern pucker,which causes the duplex to favor the A-form geometry. In addition, the2′ hydroxyl groups of RNA can form a network of water mediated hydrogenbonds that help stabilize the RNA duplex (Egli et al., Biochemistry,1996, 35, 8489-8494). On the other hand, deoxy nucleic acids prefer aC2′ endo sugar pucker, i.e., also known as Southern pucker, which isthought to impart a less stable B-form geometry (Sanger, W. (1984)Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.).

The relative ability of a chemically-modified oligomeric compound tobind to complementary nucleic acid strands, as compared to naturaloligonucleotides, is measured by obtaining the melting temperature of ahybridization complex of said chemically-modified oligomeric compoundwith its complementary unmodified target nucleic acid. The meltingtemperature (T_(m)), a characteristic physical property of doublehelixes, denotes the temperature in degrees centigrade at which 50%helical versus coiled (unhybridized) forms are present. T_(m) (alsocommonly referred to as binding affinity) is measured by using the UVspectrum to determine the formation and breakdown (melting) ofhybridization. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently a reduction in UV absorption indicates a higher T_(m).

It is known in the art that the relative duplex stability of anantisense compound:RNA target duplex can be modulated throughincorporation of chemically-modified nucleosides into the antisensecompound. Sugar-modified nucleosides have provided the most efficientmeans of modulating the T_(m) of an antisense compound with its targetRNA. Sugar-modified nucleosides that increase the population of or lockthe sugar in the C3′-endo (Northern, RNA-like sugar pucker)configuration have predominantly provided a per modification T_(m)increase for antisense compounds toward a complementary RNA target.Sugar-modified nucleosides that increase the population of or lock thesugar in the C2′-endo (Southern, DNA-like sugar pucker) configurationpredominantly provide a per modification Tm decrease for antisensecompounds toward a complementary RNA target. The sugar pucker of a givensugar-modified nucleoside is not the only factor that dictates theability of the nucleoside to increase or decrease an antisensecompound's T_(m) toward complementary RNA. For example, thesugar-modified nucleoside tricycloDNA is predominantly in the C2′-endoconformation, however it imparts a 1.9 to 3° C. per modificationincrease in T_(m) toward a complementary RNA. Another example of asugar-modified high-affinity nucleoside that does not adopt the C3′-endoconformation is α-L-LNA (described in more detail herein).

As used herein, “T_(m)” means melting temperature which is thetemperature at which the two strands of a duplex nucleic acid separate.T_(m) is often used as a measure of duplex stability or the bindingaffinity of an antisense compound toward a complementary strand such asan RNA molecule.

As used herein, “complementarity” in reference to nucleobases refers toa nucleobase that is capable of base pairing with another nucleobase.For example, in DNA, adenine (A) is complementary to thymine (T). Forexample, in RNA, adenine (A) is complementary to uracil (U). In certainembodiments, complementary nucleobase refers to a nucleobase of anantisense compound that is capable of base pairing with a nucleobase ofits target nucleic acid. For example, if a nucleobase at a certainposition of an antisense compound is capable of hydrogen bonding with anucleobase at a certain position of a target nucleic acid, then theposition of hydrogen bonding between the oligonucleotide and the targetnucleic acid is considered to be complementary at that nucleobase pair.Nucleobases or more broadly, heterocyclic base moieties, comprisingcertain modifications may maintain the ability to pair with acounterpart nucleobase and thus, are still capable of complementarity.

As used herein, “non-complementary” “in reference to nucleobases refersto a pair of nucleobases that do not form hydrogen bonds with oneanother or otherwise support hybridization.

As used herein, “complementary” in reference to linked nucleosides,oligonucleotides, oligomeric compounds, or nucleic acids, refers to thecapacity of an oligomeric compound to hybridize to another oligomericcompound or nucleic acid through nucleobase or more broadly,heterocyclic base, complementarity. In certain embodiments, an antisensecompound and its target are complementary to each other when asufficient number of corresponding positions in each molecule areoccupied by nucleobases that can bond with each other to allow stableassociation between the antisense compound and the target. One skilledin the art recognizes that the inclusion of mismatches is possiblewithout eliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are antisense compounds thatmay comprise up to about 20% nucleotides that are mismatched (i.e., arenot nucleobase complementary to the corresponding nucleotides of thetarget). Preferably the antisense compounds contain no more than about15%, more preferably not more than about 10%, most preferably not morethan 5% or no mismatches. The remaining nucleotides are nucleobasecomplementary or otherwise do not disrupt hybridization (e.g., universalbases). One of ordinary skill in the art would recognize the compoundsprovided herein are at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%complementary to a target nucleic acid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligomeric compound mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). In certain embodiments, oligomericcompounds can comprise at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, or at least about 99% sequencecomplementarity to a target region within the target nucleic acidsequence to which they are targeted. For example, an oligomeric compoundin which 18 of 20 nucleobases of the oligomeric compound arecomplementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity. In this example,the remaining noncomplementary nucleobases may be clustered orinterspersed with complementary nucleobases and need not be contiguousto each other or to complementary nucleobases. As such, an oligomericcompound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin this scope. Percent complementarity of an oligomeric compoundwith a region of a target nucleic acid can be determined routinely usingBLAST programs (basic local alignment search tools) and PowerBLASTprograms known in the art (Altschul et al., J. Mol. Biol., 1990, 215,403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

As used herein, “hybridization” refers to the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases). For example,the natural base adenine is nucleobase complementary to the naturalnucleobases thymidine and uracil which pair through the formation ofhydrogen bonds. The natural base guanine is nucleobase complementary tothe natural bases cytosine and 5-methyl cytosine. Hybridization canoccur under varying circumstances.

As used herein, “target nucleic acid” refers to any nucleic acidmolecule the expression, amount, or activity of which is capable ofbeing modulated by an antisense compound. In certain embodiments, thetarget nucleic acid is DNA or RNA. In certain embodiments, the targetRNA is mRNA, pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA,mature microRNA, promoter-directed RNA, or natural antisensetranscripts. For example, the target nucleic acid can be a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. In certain embodiments, target nucleic acid is aviral or bacterial nucleic acid.

Further included herein are oligomeric compounds such as antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these oligomeric compoundsmay be introduced in the form of single-stranded, double-stranded,circular or hairpin oligomeric compounds and may contain structuralelements such as internal or terminal bulges or loops. Once introducedto a system, the oligomeric compounds provided herein may elicit theaction of one or more enzymes or structural proteins to effectmodification of the target nucleic acid. Alternatively, the oligomericcompound may inhibit the activity the target nucleic acid through anoccupancy-based method, thus interfering with the activity of the targetnucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded oligomeric compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While one form of oligomeric compound is a single-stranded antisenseoligonucleotide, in many species the introduction of double-strandedstructures, such as double-stranded RNA (dsRNA) molecules, has beenshown to induce potent and specific antisense-mediated reduction of thefunction of a gene or its associated gene products. This phenomenonoccurs in both plants and animals and is believed to have anevolutionary connection to viral defense and transposon silencing.

As used herein, “modulation” refers to a perturbation of amount orquality of a function or activity when compared to the function oractivity prior to modulation. For example, modulation includes thechange, either an increase (stimulation or induction) or a decrease(inhibition or reduction) in gene expression. As a further example,modulation of expression can include perturbing splice site selection ofpre-mRNA processing, resulting in a change in the amount of a particularsplice-variant present compared to conditions that were not perturbed.As a further example, modulation includes perturbing translation of aprotein.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the desired activity of the compound and do not impartundesired toxicological effects thereto. The term “pharmaceuticallyacceptable salt” includes a salt prepared from pharmaceuticallyacceptable non-toxic acids or bases, including inorganic or organicacids and bases.

Pharmaceutically acceptable salts of the oligomeric compounds describedherein may be prepared by methods well-known in the art. For a review ofpharmaceutically acceptable salts, see Stahl and Wermuth, Handbook ofPharmaceutical Salts: Properties, Selection and Use (Wiley-VCH,Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides areuseful and are well accepted for therapeutic administration to humans.Accordingly, in one embodiment the oligomeric compounds described hereinare in the form of a sodium salt.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 8 to about 80 monomer subunits in length. One having ordinaryskill in the art will appreciate that this embodies oligomeric compoundsof 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,or 80 monomer subunits in length, or any range therewithin.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 8 to 40 monomer subunits in length. One having ordinary skillin the art will appreciate that this embodies oligomeric compounds of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 monomersubunits in length, or any range therewithin.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 8 to 20 monomer subunits in length. One having ordinary skillin the art will appreciate that this embodies oligomeric compounds of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 monomer subunits inlength, or any range therewithin.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 8 to 16 monomer subunits in length. One having ordinary skillin the art will appreciate that this embodies oligomeric compounds of 8,9, 10, 11, 12, 13, 14, 15 or 16 monomer subunits in length, or any rangetherewithin.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 10 to 14 monomer subunits in length. One having ordinaryskill in the art will appreciate that this embodies oligomeric compoundsof 10, 11, 12, 13 or 14 monomer subunits in length, or any rangetherewithin.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 10 to 18 monomer subunits in length. One having ordinaryskill in the art will appreciate that this embodies oligomeric compoundsof 10, 11, 12, 13, 14, 15, 16, 17 or 18 monomer subunits in length, orany range therewithin.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 10 to 21 monomer subunits in length. One having ordinaryskill in the art will appreciate that this embodies oligomeric compoundsof 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 monomer subunits inlength, or any range therewithin.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 12 to 14 monomer subunits in length. One having ordinaryskill in the art will appreciate that this embodies oligomeric compoundsof 12, 13 or 14 monomer subunits in length, or any range therewithin.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 12 to 18 monomer subunits in length. One having ordinaryskill in the art will appreciate that this embodies oligomeric compoundsof 12, 13, 14, 15, 16, 17 or 18 monomer subunits in length, or any rangetherewithin.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 12 to 21 monomer subunits in length. One having ordinaryskill in the art will appreciate that this embodies oligomeric compoundsof 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 monomer subunits in length,or any range therewithin.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 14 to 18 monomer subunits in length. One having ordinaryskill in the art will appreciate that this embodies oligomeric compoundsof 14, 15, 16, 17 or 18 monomer subunits in length, or any rangetherewithin.

In certain embodiments, oligomeric compounds of any of a variety ofranges of lengths of linked monomer subunits are provided. In certainembodiments, oligomeric compounds are provided consisting of X-Y linkedmonomer subunits, where X and Y are each independently selected from 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, and 50; provided that X<Y. For example, in certainembodiments, this provides oligomeric compounds comprising: 8-9, 8-10,8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22,8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13,9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25,9-26, 9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16,10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 10-25, 10-26,10-27, 10-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17,11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27,11-28, 11-29, 11-30, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19,12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29,12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22,13-23, 13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16,14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26,14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21,15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17,16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 16-26, 16-27,16-28, 16-29, 16-30, 17-18, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24,17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 18-19, 18-20, 18-21, 18-22,18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 19-20, 19-21,19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 20-21,20-22, 20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22,21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22-24,22-25, 22-26, 22-27, 22-28, 22-29, 22-30, 23-24, 23-25, 23-26, 23-27,23-28, 23-29, 23-30, 24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 25-26,25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29,27-30, 28-29, 28-30, or 29-30 linked monomer subunits.

In certain embodiments, the ranges for the oligomeric compounds listedherein are meant to limit the number of monomer subunits in theoligomeric compounds, however such oligomeric compounds may furtherinclude 5′ and/or 3′-terminal groups including but not limited toprotecting groups such as hydroxyl protecting groups, optionally linkedconjugate groups and/or other substituent groups.

In certain embodiments, the preparation of oligomeric compounds asdisclosed herein is performed according to literature procedures forDNA: Protocols for Oligonucleotides and Analogs, Agrawal, Ed., HumanaPress, 1993, and/or RNA: Scaringe, Methods, 2001, 23, 206-217; Gait etal., Applications of Chemically synthesized RNA in RNA:ProteinInteractions, Smith, Ed., 1998, 1-36; Gallo et al., Tetrahedron, 2001,57, 5707-5713. Additional methods for solid-phase synthesis may be foundin Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777;4,973,679; and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re.34,069.

Oligomeric compounds are routinely prepared using solid support methodsas opposed to solution phase methods. Commercially available equipmentcommonly used for the preparation of oligomeric compounds that utilizethe solid support method is sold by several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Any other means forsuch synthesis known in the art may additionally or alternatively beemployed. Suitable solid phase techniques, including automated synthesistechniques, are described in Oligonucleotides and Analogues, a PracticalApproach, F. Eckstein, Ed., Oxford University Press, New York, 1991.

The synthesis of RNA and related analogs relative to the synthesis ofDNA and related analogs has been increasing as efforts in RNAinterference and micro RNA increase. The primary RNA synthesisstrategies that are presently being used commercially include5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS),5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl](FPMP),2′-O—[(triisopropylsilyl)oxy]methyl (2′-O—CH₂—O—Si(iPr)₃ (TOM) and the5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list ofsome of the major companies currently offering RNA products includePierce Nucleic Acid Technologies, Dharmacon Research Inc., AmeriBiotechnologies Inc., and Integrated DNA Technologies, Inc. One company,Princeton Separations, is marketing an RNA synthesis activatoradvertised to reduce coupling times especially with TOM and TBDMSchemistries. The primary groups being used for commercial RNA synthesisare: TBDMS: 5′-O-DMT-2′-O-t-butyldimethylsilyl; TOM:2′-O—[(triisopropylsilyl)oxy]methyl; DOD/ACE:(5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether-2′-O-bis(2-acetoxyethoxy)methyl; and FPMP:5′-O-DMT-2′-O[1(2-fluorophenyl)-4-ethoxypiperidin-4-yl]. In certainembodiments, each of the aforementioned RNA synthesis strategies can beused herein. In certain embodiments, the aforementioned RNA synthesisstrategies can be performed together in a hybrid fashion e.g. using a5′-protecting group from one strategy with a 2′-O-protecting fromanother strategy.

In some embodiments, “suitable target segments” may be employed in ascreen for additional oligomeric compounds that modulate the expressionof a selected protein. “Modulators” are those oligomeric compounds thatdecrease or increase the expression of a nucleic acid molecule encodinga protein and which comprise at least an 8-nucleobase portion which iscomplementary to a suitable target segment. The screening methodcomprises the steps of contacting a suitable target segment of a nucleicacid molecule encoding a protein with one or more candidate modulators,and selecting for one or more candidate modulators which decrease orincrease the expression of a nucleic acid molecule encoding a protein.Once it is shown that the candidate modulator or modulators are capableof modulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding a peptide, the modulator may then beemployed herein in further investigative studies of the function of thepeptide, or for use as a research, diagnostic, or therapeutic agent. Inthe case of oligomeric compounds targeted to microRNA, candidatemodulators may be evaluated by the extent to which they increase theexpression of a microRNA target RNA or protein (as interference with theactivity of a microRNA will result in the increased expression of one ormore targets of the microRNA).

As used herein, “expression” refers to the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, splicing, post-transcriptional modification, andtranslation.

Suitable target segments may also be combined with their respectivecomplementary oligomeric compounds provided herein to form stabilizeddouble-stranded (duplexed) oligonucleotides. Such double strandedoligonucleotide moieties have been shown in the art to modulate targetexpression and regulate translation as well as RNA processing via anantisense mechanism. Moreover, the double-stranded moieties may besubject to chemical modifications (Fire et al., Nature, 1998, 391,806-811; Timmons and Fire, Nature, 1998, 395, 854; Timmons et al., Gene,2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431;Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507;Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature,2001, 411, 494-498; Elbashir et al., Genes Dev., 2001, 15, 188-200). Forexample, such double-stranded moieties have been shown to inhibit thetarget by the classical hybridization of antisense strand of the duplexto the target, thereby triggering enzymatic degradation of the target(Tijsterman et al., Science, 2002, 295, 694-697).

The oligomeric compounds provided herein can also be applied in theareas of drug discovery and target validation. In certain embodiments,provided herein is the use of the oligomeric compounds and targetsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between proteins and a disease state, phenotype, orcondition. These methods include detecting or modulating a targetpeptide comprising contacting a sample, tissue, cell, or organism withone or more oligomeric compounds provided herein, measuring the nucleicacid or protein level of the target and/or a related phenotypic orchemical endpoint at some time after treatment, and optionally comparingthe measured value to a non-treated sample or sample treated with afurther oligomeric compound as provided herein. These methods can alsobe performed in parallel or in combination with other experiments todetermine the function of unknown genes for the process of targetvalidation or to determine the validity of a particular gene product asa target for treatment or prevention of a particular disease, condition,or phenotype. In certain embodiments, oligomeric compounds are providedfor use in therapy. In certain embodiments, the therapy is reducingtarget messenger RNA.

As used herein, the term “dose” refers to a specified quantity of apharmaceutical agent provided in a single administration. In certainembodiments, a dose may be administered in two or more boluses, tablets,or injections. For example, in certain embodiments, where subcutaneousadministration is desired, the desired dose requires a volume not easilyaccommodated by a single injection. In such embodiments, two or moreinjections may be used to achieve the desired dose. In certainembodiments, a dose may be administered in two or more injections tominimize injection site reaction in an individual.

In certain embodiments, chemically-modified oligomeric compounds areprovided herein that may have a higher affinity for target RNAs thandoes non-modified DNA. In certain such embodiments, higher affinity inturn provides increased potency allowing for the administration of lowerdoses of such compounds, reduced potential for toxicity, improvement intherapeutic index and decreased overall cost of therapy.

Effect of nucleoside modifications on RNAi activity is evaluatedaccording to existing literature (Elbashir et al., Nature, 2001, 411,494-498; Nishikura et al., Cell, 2001, 107, 415-416; and Bass et al.,Cell, 2000, 101, 235-238.)

In certain embodiments, oligomeric compounds provided herein can beutilized for diagnostics, therapeutics, prophylaxis and as researchreagents and kits. Furthermore, antisense oligonucleotides, which areable to inhibit gene expression with exquisite specificity, are oftenused by those of ordinary skill to elucidate the function of particulargenes or to distinguish between functions of various members of abiological pathway. In certain embodiments, oligomeric compoundsprovided herein can be utilized either alone or in combination withother oligomeric compounds or other therapeutics as tools indifferential and/or combinatorial analyses to elucidate expressionpatterns of a portion or the entire complement of genes expressed withincells and tissues. Oligomeric compounds can also be effectively used asprimers and probes under conditions favoring gene amplification ordetection, respectively. These primers and probes are useful in methodsrequiring the specific detection of nucleic acid molecules encodingproteins and in the amplification of the nucleic acid molecules fordetection or for use in further studies. Hybridization of oligomericcompounds as provided herein, particularly the primers and probes, witha nucleic acid can be detected by means known in the art. Such means mayinclude conjugation of an enzyme to the oligonucleotide, radiolabellingof the oligonucleotide or any other suitable detection means. Kits usingsuch detection means for detecting the level of selected proteins in asample may also be prepared.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more of the oligomeric compounds provided herein arecompared to control cells or tissues not treated with oligomericcompounds and the patterns produced are analyzed for differential levelsof gene expression as they pertain, for example, to disease association,signaling pathway, cellular localization, expression level, size,structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds and or oligomeric compounds which affectexpression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.USA, 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999,20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

Those skilled in the art, having possession of the present disclosurewill be able to prepare oligomeric compounds, comprising a contiguoussequence of linked monomer subunits, of essentially any viable length topractice the methods disclosed herein. Such oligomeric compounds willinclude at least one and preferably a plurality of the 2′-thio bicyclicnucleosides provided herein and may also include other monomer subunitsincluding but not limited to nucleosides, modified nucleosides andnucleosides comprising sugar surrogate groups.

While in certain embodiments, oligomeric compounds provided herein canbe utilized as described, the following examples serve only toillustrate and are not intended to be limiting.

EXAMPLES General

¹H and ¹³C NMR spectra were recorded on a 300 MHz and 75 MHz Brukerspectrometer, respectively.

Example 1 Synthesis of Nucleoside Phosphoramidites

The preparation of nucleoside phosphoramidites is performed followingprocedures that are illustrated herein and in the art such as but notlimited to U.S. Pat. No. 6,426,220 and published PCT WO 02/36743.

Example 2 Synthesis of Oligomeric Compounds

The oligomeric compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as alkylatedderivatives and those having phosphorothioate linkages.

Oligomeric compounds: Unsubstituted and substituted phosphodiester (P═O)oligomeric compounds, including without limitation, oligonucleotides canbe synthesized on an automated DNA synthesizer (Applied Biosystems model394) using standard phosphoramidite chemistry with oxidation by iodine.

In certain embodiments, phosphorothioate internucleoside linkages (P═S)are synthesized similar to phosphodiester internucleoside linkages withthe following exceptions: thiation is effected by utilizing a 10% w/vsolution of 3-H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile forthe oxidation of the phosphite linkages. The thiation reaction step timeis increased to 180 sec and preceded by the normal capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (12-16 hr), the oligomeric compounds are recoveredby precipitating with greater than 3 volumes of ethanol from a 1 MNH₄OAc solution. Phosphinate internucleoside linkages can be prepared asdescribed in U.S. Pat. No. 5,508,270.

Alkyl phosphonate internucleoside linkages can be prepared as describedin U.S. Pat. No. 4,469,863.

3′-Deoxy-3′-methylene phosphonate internucleoside linkages can beprepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.

Phosphoramidite internucleoside linkages can be prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.

Alkylphosphonothioate internucleoside linkages can be prepared asdescribed in published PCT applications PCT/US94/00902 andPCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate internucleoside linkages can beprepared as described in U.S. Pat. No. 5,476,925.

Phosphotriester internucleoside linkages can be prepared as described inU.S. Pat. No. 5,023,243.

Borano phosphate internucleoside linkages can be prepared as describedin U.S. Pat. Nos. 5,130,302 and 5,177,198.

Oligomeric compounds having one or more non-phosphorus containinginternucleoside linkages including without limitationmethylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides,methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone oligomeric compounds having, for instance,alternating MMI and P═O or P═S linkages can be prepared as described inU.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.

Formacetal and thioformacetal internucleoside linkages can be preparedas described in U.S. Pat. Nos. 5,264,562 and 5,264,564.

Ethylene oxide internucleoside linkages can be prepared as described inU.S. Pat. No. 5,223,618.

Example 3 Isolation and Purification of Oligomeric Compounds

After cleavage from the controlled pore glass solid support or othersupport medium and deblocking in concentrated ammonium hydroxide at 55°C. for 12-16 hours, the oligomeric compounds, including withoutlimitation oligonucleotides and oligonucleosides, are recovered byprecipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligomeric compounds are analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresis.The relative amounts of phosphorothioate and phosphodiester linkagesobtained in the synthesis is determined by the ratio of correctmolecular weight relative to the 16 amu product (+/−32+/−48). For somestudies oligomeric compounds are purified by HPLC, as described byChiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtainedwith HPLC-purified material are generally similar to those obtained withnon-HPLC purified material.

Example 4 Synthesis of Oligomeric Compounds Using the 96 Well PlateFormat

Oligomeric compounds, including without limitation oligonucleotides, canbe synthesized via solid phase P(III) phosphoramidite chemistry on anautomated synthesizer capable of assembling 96 sequences simultaneouslyin a 96-well format. Phosphodiester internucleoside linkages areafforded by oxidation with aqueous iodine. Phosphorothioateinternucleoside linkages are generated by sulfurization utilizing3-H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrousacetonitrile. Standard base-protected beta-cyanoethyl-diiso-propylphosphoramidites can be purchased from commercial vendors (e.g.PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway,N.J.). Non-standard nucleosides are synthesized as per standard orpatented methods and can be functionalized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligomeric compounds can be cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product is thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 5 Analysis of Oligomeric Compounds Using the 96-Well PlateFormat

The concentration of oligomeric compounds in each well can be assessedby dilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products can be evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition isconfirmed by mass analysis of the oligomeric compounds utilizingelectrospray-mass spectroscopy. All assay test plates are diluted fromthe master plate using single and multi-channel robotic pipettors.Plates are judged to be acceptable if at least 85% of the oligomericcompounds on the plate are at least 85% full length.

Example 6 In Vitro Treatment of Cells with Oligomeric Compounds

The effect of oligomeric compounds on target nucleic acid expression istested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Cell linesderived from multiple tissues and species can be obtained from AmericanType Culture Collection (ATCC, Manassas, Va.).

The following cell type is provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays or RT-PCR.

b.END cells: The mouse brain endothelial cell line b.END was obtainedfrom Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany).b.END cells are routinely cultured in DMEM, high glucose (InvitrogenLife Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells areroutinely passaged by trypsinization and dilution when they reachedapproximately 90% confluence. Cells are seeded into 96-well plates(Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a densityof approximately 3000 cells/well for uses including but not limited tooligomeric compound transfection experiments.

Experiments involving treatment of cells with oligomeric compounds:

When cells reach appropriate confluency, they are treated witholigomeric compounds using a transfection method as described.

Lipofectin™

When cells reached 65-75% confluency, they are treated with one or moreoligomeric compounds. The oligomeric compound is mixed with LIPOFECTIN™Invitrogen Life Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reducedserum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achievethe desired concentration of the oligomeric compound(s) and aLIPOFECTIN™ concentration of 2.5 or 3 μg/mL per 100 nM oligomericcompound(s). This transfection mixture is incubated at room temperaturefor approximately 0.5 hours. For cells grown in 96-well plates, wellsare washed once with 100 μL OPTI-MEM™-1 and then treated with 130 μL ofthe transfection mixture. Cells grown in 24-well plates or otherstandard tissue culture plates are treated similarly, using appropriatevolumes of medium and oligomeric compound(s). Cells are treated and dataare obtained in duplicate or triplicate. After approximately 4-7 hoursof treatment at 37° C., the medium containing the transfection mixtureis replaced with fresh culture medium. Cells are harvested 16-24 hoursafter treatment with oligomeric compound(s).

Other suitable transfection reagents known in the art include, but arenot limited to, CYTOFECTIN™, LIPOFECTAMINE™, OLIGOFECTAMINE™, andFUGENE™. Other suitable transfection methods known in the art include,but are not limited to, electroporation.

Example 7 Real-Time Quantitative PCR Analysis of Target mRNA Levels

Quantitation of target mRNA levels is accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

RT and PCR reagents are obtained from Invitrogen Life Technologies(Carlsbad, Calif.). RT, real-time PCR is carried out by adding 20 μL PCRcocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP,dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer,125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction iscarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM° Taq, 40 cycles of atwo-step PCR protocol are carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by RT, real-time PCR are normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RIBOGREEN™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RIBOGREEN™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RIBOGREEN™ working reagent (RIBOGREEN™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Example 8 Analysis of Oligonucleotide Inhibition of Target Expression

Antisense modulation of a target expression can be assayed in a varietyof ways known in the art. For example, a target mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR. Real-time quantitative PCR ispresently desired. RNA analysis can be performed on total cellular RNAor poly(A)+ mRNA. One method of RNA analysis of the present disclosureis the use of total cellular RNA as described in other examples herein.Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISM™7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Protein levels of a target can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art. Methods for preparation ofpolyclonal antisera are taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9,John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons,Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 9 Design of Phenotypic Assays and In Vivo Studies for the Use ofTarget Inhibitors Phenotypic Assays

Once target inhibitors have been identified by the methods disclosedherein, the oligomeric compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of a target in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with atarget inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Measurement of the expression of one or more of the genes of the cellafter treatment is also used as an indicator of the efficacy or potencyof the target inhibitors. Hallmark genes, or those genes suspected to beassociated with a specific disease state, condition, or phenotype, aremeasured in both treated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

Example 10 RNA Isolation

Poly(A)+mRNA Isolation

Poly(A)+mRNA is isolated according to Miura et al., (Clin. Chem., 1996,42, 1758-1764). Other methods for poly(A)+mRNA isolation are routine inthe art. Briefly, for cells grown on 96-well plates, growth medium isremoved from the cells and each well is washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) is added to each well, theplate is gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate is transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates are incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plateis blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C., is added to each well, the plate is incubated on a90° C. hot plate for 5 minutes, and the eluate is then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA is isolated using an RNEASY 96™ kit and buffers purchased fromQiagen Inc. (Valencia, Calif.) following the manufacturer's recommendedprocedures. Briefly, for cells grown on 96-well plates, growth medium isremoved from the cells and each well is washed with 200 μL cold PBS. 150μL Buffer RLT is added to each well and the plate vigorously agitatedfor 20 seconds. 150 μL of 70% ethanol is then added to each well and thecontents mixed by pipetting three times up and down. The samples arethen transferred to the RNEASY 96™ well plate attached to a QIAVAC™manifold fitted with a waste collection tray and attached to a vacuumsource. Vacuum is applied for 1 minute. 500 μL of Buffer RW1 is added toeach well of the RNEASY 96™ plate and incubated for 15 minutes and thevacuum is again applied for 1 minute. An additional 500 μL of Buffer RW1is added to each well of the RNEASY 96™ plate and the vacuum is appliedfor 2 minutes. 1 mL of Buffer RPE is then added to each well of theRNEASY 96™ plate and the vacuum applied for a period of 90 seconds. TheBuffer RPE wash is then repeated and the vacuum is applied for anadditional 3 minutes. The plate is then removed from the QIAVAC™manifold and blotted dry on paper towels. The plate is then re-attachedto the QIAVAC™ manifold fitted with a collection tube rack containing1.2 mL collection tubes. RNA is then eluted by pipetting 140 μL of RNAsefree water into each well, incubating 1 minute, and then applying thevacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 11 Target-Specific Primers and Probes

Probes and primers may be designed to hybridize to a target sequence,using published sequence information.

For example, for human PTEN, the following primer-probe set was designedusing published sequence information (GENBANK™ accession numberU92436.1, SEQ ID NO: 1).

Forward primer: AATGGCTAAGTGAAGATGACAATCAT (SEQ ID NO: 2)Reverse primer: TGCACATATCATTACACCAGTTCGT (SEQ ID NO: 3)And the PCR probe:

FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 4), where FAM isthe fluorescent dye and TAMRA is the quencher dye.

Example 12 Western Blot Analysis of Target Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to a target is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 13 Preparation of Compound 7

a) Preparation of Compound 2

Commercially available 1,2;5,6-di-O-isopropylidene-α-D-allofuranose,Compound 1, (135 g, 519.0 mmol) and 2-(bromomethyl)-naphthalene (126 g,570.0 mmol) were dissolved in DMF (500 mL) in a three-necked flask (500mL) and the reaction was cooled in an ice bath. Sodium hydride (60% w/w,29 g, 727.0 mmol) was carefully added (6 g portions every 10 minutes) tothe reaction and the stirring was continued for another 60 minutes afterthe addition was complete. At this time TLC analysis showed no moresugar (Compound 1). The reaction was carefully poured onto crushed ice(ca. 500 g) and the resulting slurry was stirred vigorously until allthe ice melted. The resulting off-white solid was collected byfiltration and suspended in water. The suspension was stirred vigorouslyusing a mechanical stirrer for 30 minutes after which the solid wascollected by filtration and suspended in hexanes. The suspension wasstirred vigorously for 30 minutes after which the solid was collected byfiltration and air dried for 4-6 hours and then dried under high vacuumover P₂O₅ for 16 hours to provide Compound 2 (206.0 g, 99%) as anoff-white solid. ¹H NMR (300 MHz, CDCl₃) δ: 7.85 (m, 4H), 7.48 (m, 3H),5.74 (s, 1H), 4.92 (d, 1H, J=11.7), 4.75 (d, 1H, J=11.6), 4.58 (m, 1H),4.36 (m, 1H), 4.15 (m, 1H), 4.03-3.86 (m, 3H), 1.61 (s, 3H), 1.36 (s,9H).

b) Preparation of Compound 3

Compound 2 (200.0 g, 0.5 moles) was added in small portions to asolution of acetic acid (2.2 L) and water (740 mL). The reaction wasstirred at room temperature for 16 h after which, TLC analysis (30%EtOAc/hexanes) indicated complete consumption of Compound 2. Thereaction was then concentrated under reduced pressure until most of theacetic acid was removed. The remaining solution was poured into astirred mixture of EtOAc (1 L) and water (1 L). Solid KOH was then addedto the above mixture until the aqueous layer was strongly basic (pH>12).The organic layer was then separated, washed with saturated sodiumbicarbonate solution and brine then dried (Na₂SO₄), filtered andconcentrated under reduced pressure to provide Compound 3 as a yellowfoam, which was used without any further purification.

c) Preparation of Compound 4

A solution of NaIO₄ (107.0 g) in water (3 L) was added over 40 minutesto a stirred (mechanical stirrer) solution of Compound 3 (crude fromabove) in dioxane (1.5 L). After 60 minutes the reaction mixture waspoured into EtOAc (1.5 L) and the organic layer was separated, washedwith water (1 L) and brine (1 L) then dried (Na₂SO₄) and concentrated toprovide Compound 4 as a yellow oil, which was used without any furtherpurification.

d) Preparation of Compound 5

Compound 4 (crude from above) was dissolved in a mixture of THF (500)and water (500 mL) and the reaction was cooled in an ice bath. 2N NaOH(600 mL) and formaldehyde (250 mL of a 37% aqueous solution) were addedto the reaction with stirring at room temperature for 3 days. Thereaction was then poured into EtOAc (1 L) and washed with water (1 L)and brine (1 L) then evaporated under reduced pressure untilapproximately 200 mL of EtOAc was left (a white precipitate was formedin the process). Hexanes (300 mL) was added to the precipitate and themixture was allowed to stand for 16 hours after which the white solidwas collected by filtration, washed with hexanes and dried under highvacuum over P₂O₅ to provide Compound 5 as a white solid (124 g, 66% fromCompound 3). ¹H NMR (300 MHz, CDCl₃) δ: 7.85 (m, 4H), 7.48 (m, 3H), 5.75(d, 1H, J=3.9), 4.96 (d, 1H. J=11.8), 4.75 (d, 1H, J=11.8), 4.66 (m,1H), 4.26 (d, 1H, J=5.2), 3.95 (m, 2H), 3.79 (m, 1H), 3.63 (m, 1H), 2.39(m, 1H, OH), 1.66 (s, 3H), 1.34 (s, 3H).

e) Preparation of Compounds 6 and 7

tert-Butyldiphenylchlorosilane (305.0 mmol, 84.0 mL) was added to a cold(0° C.) stirring solution of Compound 5 (278.0 mmol, 100.0 g) andtriethylamine (305 mmol, 43.0 mL) in dichloromethane (600 mL). After theaddition was complete, the reaction was warmed to room temperature andthe stirring was continued for 16 hours. MeOH (50 mL) was added (toquench the excess TBDPSCl) to the reaction and the stirring wascontinued for another 2 hours at room temperature. The reaction was thendiluted with chloroform and the organic layer was washed with 10% HCl,saturated NaHCO₃, brine, dried (Na₂SO₄) and concentrated to provide athick oil. Hexanes (150 mL) were added to the oil and the mixture wassonicated until a solution resulted. The solution was seeded with asmall amount of Compound 6 (previously isolated by columnchromatography). After standing for 16 hours additional hexanes wereadded to the thick slurry and the solid was collected by filtration. Thesolid was then resuspended in hexanes and stirred vigorously for 30minutes. The solid was collected by filtration to provide Compound 6(80.5, 48% g) after drying under high vacuum for 16 hours. The filtrateswere combined and concentrated under reduced pressure. The resulting oilwas redissolved in minimum amount of hexanes and purified by silica gelcolumn chromatography (gradient of up to 20% EtOAc in hexanes).Fractions containing Compound 7 were combined and concentrated toprovide purified Compound 7.

Compound 6 ¹H NMR (300 MHz, CDCl₃) δ: 7.83 (m, 4H), 7.56 (m, 7H), 7.30(m, 6H), 5.80 (s, 1H), 4.97 (d, 1H, J=11.4), 4.70 (m, 2H), 4.46 (m, 1H),3.92-3.66 (m, 4H), 2.39 (m, 1H, OH), 1.67 (s, 3H), 1.37 (s, 3H), 0.92(s, 9H).

Compound 7 ¹H NMR (300 MHz, CDCl₃) δ: 7.9-7.3 (m, 17H), 5.71 (d, 1H,J=3.9), 4.86 (d, 1H, J=12.2), 4.74 (d, 1H, J=12.2), 4.56 (m, 1H), 4.22(d, 1H, J=11.1), 4.18 (m, 1H), 4.07 (d, 1H, J=11.1), 4.02 (dd, 1H,J=4.2, 12.0), 3.64 (dd, 1H, J=9.4, 11.9), 1.89 (m, 1H), 1.25 (s, 6H),1.05 (s, 9H).

Example 14 Preparation of Compound 12

a) Preparation of Compound 8

Compound 6 was prepared as per the procedures illustrated in Example 13.Concentrated H₂SO₄ (2 drops) was added to a solution of Compound 6 (18g, 30.06 mmol) in glacial acetic acid (88 mL) and acetic anhydride (22mL). After stirring at room temperature for 2 hour, the reaction mixturewas poured into ethyl acetate (300 mL) and the organic layer was washedwith water (200 mL), saturated NaHCO₃ (200 mL), brine (200 mL), dried(Na₂SO₄) and concentrated under reduced pressure. Purification by columnchromatography (SiO₂, eluting with 20% to 33% ethyl acetate/hexanes)provided Compound 8 (18.53 g, 90%, a/(3 mixture, 4:1). ¹H NMR (Majorisomer, 300 MHz, CDCl₃) δ 7.85-7.31 (m, 17H), 6.19 (s, 1H), 7.30 (m,6H), 5.43 (d, J=5.1 Hz, 1H), 4.978-4.68 (m, 2H), 4.56-4.51 (m, 3H), 3.68(m, 2H), 2.12 (s, 3H), 1.96 (s, 3H), 1.04 (s, overlapping with otherisomer, 9H,); MS (ES) m/z 707.1 [M+Na]⁺.

b) Preparation of Compound 9

Compound 8 (18 g, 26.30 mmol) was mixed with uracil (5.90 g, 52.6 mmol)and dried over P₂O₅ under reduced pressure over night. The reactionmixture was suspended in anhydrous CH₃CN (113 mL) andN,O-Bis(trimethylsilyl)acetamide (38.58 mL, 117.80 mmol) added. Afterheating at 67° C. for 1.5 h to get a clear solution, the reactionmixture was cooled to 0° C. To this trimethylsilyl triflate (9.52 mL,52.60 mmol) was added. The reaction mixture was stirred at 0° C. for 15min then heated at 70° C. for 1.5 h. The reaction mixture was cooled toroom temperature and poured into ethyl acetate (200 mL). The organiclayer was washed with saturated NaHCO₃ (100 mL), brine (100 mL), dried(Na₂SO₄) and concentrated under reduced pressure. The residue waspurified by flash silica gel column chromatography and eluted with 5%methanol in CH₂Cl₂ to provide Compound 9 (16.85 g, 87%). ¹H NMR (300MHz, CDCl₃) δ 8.22 (brs, 1H), 7.63-7.08 (m, 18H), 5.98 (d, J=4.9 Hz,1H), 5.22 (t, J=5.3 Hz, 1H), 5.14 (d, J=7.9 Hz, 1H), 4.54 (d, J=11.5 Hz,1H), 4.38-4.26 (m, 3H), 3.87 (d, J=12.4 Hz, 1H), 3.70 (d, J=11.3 Hz,1H), 3.49 (d, J=11.3 Hz, 1H), 1.88 (s, 3H), 1.70 (s, 3H), 0.82 (s, 9H);¹³C NMR (75 MHz, CDCl₃) δ 170.6, 170.3, 162.91, 150.2, 140.2, 134.6,133.4, 132.8, 132.1, 130.5, 130.4, 128.6, 128.2, 128.0, 127.0, 126.6,126.5, 125.8, 103.1, 87.5, 87.0, 77.4, 77.1, 74.9, 65.0, 63.3, 27.2,20.9, 19.5; MS (ES) m/z 735.1 [M−]⁻.

c) Preparation of Compound 10

Compound 9 (16.7 g, 22.66) was dissolved in methanolic ammonia (7 M, 123mL). The reaction mixture was stirred at room temperature for 18 h.Solvent was removed under reduced pressure and the residue was purifiedby column chromatography and eluted with 5% methanol in dichloromethaneto yield Compound 10 (14.18 g, 96%). ¹HNMR (300 MHz, DMSO-d₆) δ 11.35(s, 1H), 7.94-7.89 (m, 4H), 7.59-7.30 (m, 14H), 5.95 (d, J=5.1 Hz, 1H),5.74 (d, J=7.4 Hz, 1H), 5.17 (d, J=7.9 Hz, 1H), 5.04 (t, J=5.9 and 5.1Hz, 1H), 4.97 (d, J=12.0 Hz, 1H), 4.69 (d, J=12.1 Hz, 1H), 4.4-4.38 (m,1H), 4.26 (d, J=5.9 Hz, 1H), 3.81-3.69 (m, 3H) 3.59-3.53 (m, 1H), 0.93(s, 9H), ¹³C NMR (75 MHz, CDCl₃) δ 163.7, 151.2, 140.3, 135.7, 135.5,134.9, 133.4, 133.3, 132.9, 132.2, 130.3, 130.2, 128.6, 128.2, 128.1,127.9, 127.2, 126.4, 126.3, 126.1, 102.7, 91.4, 89.2, 76.4, 75.0, 73.5,65.1, 63.0, 27.1, 19.5; MS (ES) m/z 650.9 [M−H]⁻.

d) Preparation of Compound 11

Compound 10 (14 g, 21.46 mmol) was dried over P₂O₅ under reducedpressure. Methanesulfonyl chloride (7.51 mL, 96.68 mmol) was added to acold (0° C.) solution of Compound 10 in anhydrous pyridine (118 mL).After stirring at room temperature for 3 h, the reaction mixture waspoured into ethyl acetate and the organic layer was sequentially washedwith saturated NaHCO₃ (400 mL), brine (400 mL), dried (Na₂SO₄) andconcentrated under vacuum. The residue obtained was purified using flashsilica gel column chromatography and eluted with 5% MeOH in CH₂Cl₂ toprovide Compound 11 (16.21 g, 93% yield). ¹H NMR (300 MHz, CDCl₃) δ 8.33(s, 1H), 7.86-7.79 (m, 4H), 7.58-7.28 (m, 14H), 6.13 (d, J=3.6 Hz, 1H),5.38 (m, 1H), 5.31 (d, J=8.1 Hz, 1H), 4.96 (d, J=11.5 Hz, 1H), 4.65 (d,J=11.3 Hz, 1H), 4.57-4.53 (m, 2H), 4.23 (d, J=11.5 Hz, 1H), 3.98 (d,J=11.3 Hz, 1H) 3.77 (d, J=11.3 Hz, 1H), 3.18 (s, 3H), 2.84 (s, 3H), 1.05(s, 9H); MS (ES) m/z 806.9 [M−H]⁻.

e) Preparation of Compound 12

To a solution of Compound 11 (16.00 g, 19.74 mmol) in anhydrous CH₃CN(135 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (5.46 mL, 39.48mmol). After stirring at room temperature for 3 h, the mixture wasdiluted with EtOAc (300 mL), washed with 1% (v/v) aqueous acetic acid(1×400 mL) and brine (2×400 mL), dried over anhydrous Na₂SO₄, filtered,and evaporated to a foam. The foam was redissolved in 1,4-dioxane (216mL) and 2 M aqueous NaOH (54 mL) was added. After 45 min, the mixturewas neutralized with AcOH, diluted in ethyl acetate (400 mL), washedwith saturated aqueous NaHCO₃ (1×300 mL) and brine (300 mL), dried overanhydrous Na₂SO₄, filtered, and evaporated. Purification by silica gelchromatography (5% MeOH in CH₂Cl₂) yielded Compound 12 (12.25 g, 84.8%yield) as a white foam. ¹H NMR (300 MHz, DMSO-d₆) δ 11.33 (s, 1H), 7.91(br s, 4H), 7.59-7.34 (m, 14H), 6.22 (d, J=4.9 Hz, 1H), 6.02 (d, J=4.7Hz, 1H), 5.16 (d, J=8.1 Hz, 1H), 4.97 (d, J=12.2 Hz, 1H), 4.79 (d,J=12.2 Hz, 1H), 4.58-4.50 (m, 2H), 4.40 (d, J=10.4 Hz, 1H), 4.34 (br s,1H) 3.84 (m, 2H), 3.16 (s, 3H), 0.85 (s, 9H); MS (ES) m/z 731.2 [M+H]⁺.

Example 15 Preparation of Compound 17

Compound 12 was prepared as per the procedures illustrated in Example14. The structure of Compound 17 was confirmed by spectral analysis, ¹HNMR and mass spectroscopy.

Example 16 Preparation of Compound 19

Compound 17 is prepared as per the procedures illustrated in Example 15.

Example 17 Preparation of Compound 23

Compound 17 was prepared as per the procedures illustrated in Example15. Compound 23 was prepared as per the procedures illustrated above(Example 17) and confirmed by spectral analysis, ¹H NMR and massspectroscopy.

Example 18 Preparation of Compound 25

a) Preparation of Compound 24

Compound 23 was prepared as per the procedures illustrated in Example17. Compound 23 (0.85 g, 1.23 mmol) was dissolved in anhydrousacetonitrile (6.1 mL) and (1S)-(+)-10-(camphorsulfonyl)oxaziridine (0.49g, 2.13 mmol) was added. The mixture was stirred at room temperature for18 h. The reaction mixture was concentrated under reduced pressure toobtain the crude residue, which was then purified by silica gel columnchromatography (50% EtOAc in hexanes) to yield Compound 24 (0.46 g,53%). ¹H NMR (300 MHz, CDCl₃): δ 13.20 (s, 1H), 8.31-7.94 (br s, 2H),7.87 (s, 1H), 7.61-7.01 (m, 13H), 6.96-6.69 (m, 4H), 6.19 (s, 1H), 4.99(s, 2H), 4.51 (s, 1H), 3.62-3.30 (m, 3H), 2.47 (d, J=1.0 Hz, 1H), 1.63(s, 3H). ES MS m/z 708.2 [M+H]⁺.

b) Preparation of Compound 25

Compound 24 (0.43 g, 0.60 mmol) was dried over P₂O₅ under reducedpressure, then dissolved in anhydrous DMF (2.6 mL). 1-H-tetrazole (0.037g, 0.53 mmol), N-methylimidazole (0.018 mL, 0.22 mmol) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.29 mL, 0.90mmol) were then added. After 3 hours, EtOAc (50 mL) was added and themixture was washed with aqueous NaHCO₃ (50 mL) and brine (50 mL) thendried over anhydrous Na₂SO₄, filtered and evaporated in vacuo to give anoil. The oily residue was purified by silica gel column chromatography(50% EtOAc in hexanes) to yield Compound 25 (0.46 g, 85%) as a whitefoam. MS (ES): m/z 906.2 [M−H]⁻. ³¹P NMR (121 MHz, CDCl₃): δ 150.9,150.8.

Example 19 Preparation of Compound 35

Compound 8 was prepared as per the procedures illustrated in Example 14.Compound 35 was prepared as per the procedures illustrated above(Example 19) and confirmed by spectral analysis, ¹H NMR and massspectroscopy. An alternative method for the preparation of Compound 27was reported by Pedersen, D. S.; Koch, T. Synthesis 2004, 4, 578-582.

Example 20 Preparation of Compound 39

Compound 33 was prepared as per the procedures illustrated in Example19. Compound 39 was prepared as per the procedures illustrated above(Example 20) and confirmed by spectral analysis, ¹H NMR and massspectroscopy.

Example 21 Preparation of Compound 41

Compound 39 is prepared as per the procedures illustrated in Example 20.

Example 22 Preparation of Compound 52

Compound 5 is prepared as per the procedures illustrated in Example 13.

Example 23 Preparation of Compound 60

Compound 44 is prepared as per the procedures illustrated in Example 22.Compound 44a, N-benzoyl Adenine is commercially available.

Example 24 Preparation of Compound 63

a) Preparation of Compound 61

Compound 6 was prepared as per the procedures illustrated in Example 13.Dimethylsulfoxide (10.8 mL, 152.0 mmol) was added dropwise to a cold(−78° C.) solution of oxalyl chloride (6.7 mL, 76.0 mmol) indichloromethane (400 mL). After stirring for 30 min, a solution ofCompound 6 (34.2 g, 56.4 mmol) in dichloromethane (40 mL) was added tothe reaction mixture. The stirring was continued for 45 min at −78° C.and triethylamine (31.4 mL, 224.0 mmol) was added. After stirring for 15min at −78° C., the ice bath was removed and the reaction was allowed togradually warm to rt over 45 min. The reaction was diluted withdichloromethane and the organic phase was sequentially washed with 5%aqueous HCl, saturated sodium bicarbonate, brine, then dried over Na₂SO₄and concentrated in vacuo to provide Compound 61, which was used withoutany further purification.

b) Preparation of Compounds 62 and 63

A suspension of cerium III chloride (9.2 g, 37.5 mmol) in THF (400 mL)was stirred at rt for 60 min. The reaction was cooled in an ice bath andmethyl magnesium bromide (75.0 mL of a 1.0 M solution in THF) was addedover 5 min. After stirring at 0° C. for 90 min, the reaction was cooledto −78° C. and a solution of crude aldehyde, Compound 61 in THF (75 mL)was added to the reaction mixture. After 3 h at −78° C., the reactionwas allowed to gradually warm to rt and carefully quenched withsaturated ammonium chloride. The reaction was diluted with ethyl acetateand the organic layer was sequentially washed with 5% HCl, saturatedsodium bicarbonate, brine, then dried over Na₂SO₄ and concentrated underreduced pressure. The resulting residue was purified using silica gelcolumn chromatography eluting with 10 to 30% ethyl acetate in hexanes toprovide the pure alcohol, Compound 62 (7.4 g, 21% from Compound 6) and amixture of Compounds 62 and 63 (26.3 g, 76% from Compound 6, Compounds62:63=10:1) was recovered as viscous oils.

Compound 62 ¹H NMR (300 MHz, CDCl₃) δ: 7.89-7.79 (m, 4H), 7.65-7.26 (m,13H), 5.84 (d, J=3.6 Hz, 1H), 5.05 (d, J=11.5 Hz, 1H), 4.83-4.53 (m,4H), 3.91 (d, J=11.1 Hz, 1H), 3.84 (d, J=11.1 Hz, 1H), 3.36 (s, 1H),1.63 (s, 3H), 1.39 (s, 3H), 1.10 (d, J=6.6 Hz, 3H), 0.91 (s, 9H). ¹³CNMR (75 MHz, CDCl₃) δ: 135.6, 135.5, 134.4, 133.3, 133.3, 133.2, 133.1,129.7, 129.7, 128.7, 128.0, 127.8, 127.7, 127.7, 127.2, 126.4, 126.3,125.7, 113.8, 104.8, 88.6, 79.4, 78.3, 73.0, 68.8, 62.4, 27.1, 26.8,26.7, 19.2, 16.1. ESI-MS m/z: [M+Na]⁺ found 635.2. calcd 635.2907.

Compound 63 ¹H NMR (300 MHz, CDCl₃) δ: 7.88-7.78 (m, 4H), 7.61-7.27 (m,13H), 5.87 (d, J=3.6 Hz, 1H), 4.96 (d, J=12.1 Hz, 1H), 4.74 (t, 1H),4.66 (d, J=12.1 Hz, 1H), 4.54 (d, J=5.3 Hz, 1H), 4.32-4.18 (m, 1H), 3.69(d, J=10.7 Hz, 1H), 3.52 (d, J=10.7 Hz, 1H), 3.12 (s, 1H), 1.69 (s, 3H),1.39 (s, 3H), 1.11 (d, J=6.4 Hz, 3H), 0.90 (s, 9H). ¹³C NMR (75 MHz,CDCl₃) δ: 135.5, 134.8, 133.2, 133.2, 132.9, 132.8, 129.8, 129.7, 128.4,127.9, 127.7, 126.9, 126.3, 126.1, 125.7, 114.3, 104.5, 90.4, 79.6,78.1, 72.8, 67.1, 64.6, 26.9, 26.7, 19.1, 17.0. ESI-MS m/z: [M+Na]⁺found 635.2. calcd 635.2907.

c) An Alternative Method in the Preparation of Compound 63

Dimethylsulfoxide (37.9 mL, 489.0 mmol) was added dropwise to a cold(−78° C.) solution of oxalyl chloride (21.4 mL, 244.0 mmol) indichloromethane (800 mL). After stirring for 30 min, a solution ofCompound 62 (100.0 g, 163.0 mmol) in dichloromethane (200 mL) was addedto the reaction mixture. The stirring was continued for 45 min at −78°C. and triethylamine (102.0 mL, 726.0 mmol) was added. After stirring at−78° C. for 15 min, the ice bath was removed and the reaction wasallowed to gradually warm to rt over 45 min. The reaction was dilutedwith dichloromethane and the organic phase was sequentially washed with10% citric acid solution, saturated sodium bicarbonate, brine, thendried over Na₂SO₄ and concentrated under reduced pressure to provide thecrude ketone, Compound 64, which was used without any furtherpurification.

A solution of lithium borohydride (122.0 mL of a 2M solution in THF, 244mmol) was added drop-wise over 30 min to a cold (−78° C.) solution ofCompound 64 (99.6 g, 163 mmol) in methanol (500 mL). After the additionwas complete, the cooling bath was removed and the reaction was stirredfor 2 h. The reaction was then cooled in an ice bath and carefullyquenched with saturated NH₄Cl solution and diluted with ethyl acetate.The organic layer was separated and sequentially washed with water,saturated sodium bicarbonate, brine, then dried over Na₂SO₄ andconcentrated under reduced pressure. The resulting residue was purifiedusing silica gel column chromatography eluting with 30% ethyl acetate inhexanes to furnish Compound 63 (97.2 g, 95%, Compounds 63:62>15:1) as aviscous oil. The spectroscopic analysis is identical to those reportedabove.

Example 25 Preparation of Compound 75

Compound 63 is prepared as per the procedures illustrated in Example 24.

Example 26 Preparation of Compound 81

Compound 73 is prepared as per the procedures illustrated in Example 25.

Example 27 Preparation of Compound 92

Compound 7 is prepared as per the procedures illustrated in Example 13.

Example 28 Preparation of Compound 97

Compound 91 is prepared as per the procedures illustrated in Example 27.

Example 29 Preparation of Compound 101

Compound 89 is prepared as per the procedures illustrated in Example 27.

Example 30 General Procedure for the Preparation of Compounds of FormulaIII

Bx is a heterocyclic base moiety;R₁, R₂, R₃ and R₄ are each independently H or a substituent group.

In certain embodiments, compounds of Formula I_(c) are prepared as perthe procedures illustrated in Examples 15, 17, 19, 20, 22, 23, and25-28. In certain embodiments, compounds of Formula I_(c) are preparedusing the procedures illustrated in the examples illustrated above andmethods and procedures known in the art.

Example 31 T_(m) Measurements

A Cary 100 Bio spectrophotometer with the Cary Win UV Thermal programwas used to measure absorbance vs. temperature. For the T_(m)experiments, oligonucleotides were prepared at a concentration of 8 μMin a buffer of 100 mM Na+, 10 mM phosphate, 0.1 mM EDTA, pH 7.Concentration of oligonucleotides were determined at 85° C. Theoligonucleotide concentration was 4 μM with mixing of equal volumes oftest oligonucleotide and match or mismatch RNA strand. Oligonucleotideswere hybridized with the complimentary or mismatch RNA strand by heatingduplex to 90° C. for 5 min and allowed to cool at room temperature.Using the spectrophotometer, T_(m) measurements were taken by heatingduplex solution at a rate of 0.5 C/min in cuvette starting @ 15° C. andheating to 85° C. T_(m) values were determined using Vant Hoffcalculations (A₂₆₀ vs temperature curve) using non self-complementarysequences where the minimum absorbance which relates to the duplex andthe maximum absorbance which relates to the non-duplex single strand aremanually integrated into the program.

Example 32 Preparation of Oligomeric Compounds

Following synthetic procedures well know in the art, some of which areillustrated herein, oligomeric compounds are prepared having at leastone substituted 2′-sulfoxide or 2′-sulfone bicyclic nucleosides, usingone or more of the phosphoramidite compounds illustrated in the Examplessuch as DMT phosphoramidites (see Compound 19, Example 16, Compound 25,Example 18, Compound 35, Example 19, Compound 41, Example 21, Compound52, Example 22, Compound 60, Example 23, Compound 75, Example 25,Compound 81, Example 26, Compound 92, Example 27, Compound 97, Example28, Compound 101, Example 20 and Compounds of Formula III, Example 30).

Example 33 T_(m) Analysis for β-D-2′-deoxyribonucleoside,4′-CH₂—O-2′-BNA, 6′-(S)—CH₃—BNA or 2′-(R)-Sulfoxide-BNA

In accordance with the present invention, oligomeric compounds weresynthesized and Tm's were assessed as illustrated in Example 31 using 4μM β-D-2′-deoxyribonucleoside, 4′-CH₂—O-2′-BNA, 6′-(S)—CH₃—BNA or2′-(R)-Sulfoxide-BNA modified oligomers and 4 μM RNA complement5′-AGCAAAAAACGC-3′ (SEQ ID NO: 5)

SEQ ID NO./ Composition Tm ΔTm/mod ISIS NO. (5′ to 3′) (° C.) (° C.)06/438705 GCGTTTTTTGCT 45.4 — 06/438707 GCGTTT₁TTTGCT 50.4 4.6 06/438709GCGTTT_(S)TTTGCT 50.5 4.7 06/462886 GCGTTT_(R)TTTGCT 49.2 3.8

Each internucleoside linkage is a phosphodiester. Italicized nucleosidesare β-D-ribonucleosides, non-italicized nucleosides not followed by asubscript are β-D-2′-deoxyribonucleosides, subscript 1 indicates thatthe preceding nucleoside is 4′-CH₂—O-2′-BNA, subscript S indicates thatthe preceding nucleoside is 6′-(S)—CH₃—BNA, subscript R indicates thatthe preceding nucleoside is 2′-(R)-Sulfoxide-BNA (structures drawnbelow). S indicates the configuration at carbon 6 position of6-(S)—CH₃—BNA. R indicates the configuration of the sulfoxide at carbon2 position of 2′-(R)-Sulfoxide-BNA.

What is claimed is:
 1. A bicyclic nucleoside having Formula Ia:

wherein: Bx is a heterocyclic base moiety; one of T₁ and T₂ is H or ahydroxyl protecting group and the other of T₁ and T₂ is H, a hydroxylprotecting group or a reactive phosphorus group; Q₁ and Q₂ are eachindependently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;G₁ and G₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl; each substituted group is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, OJ₁, SJ₁, NJ₁J₂, N₃, CN, C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁,O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ and N(H)C(═S)NJ₁J₂; each J₁ and J₂ is,independently, H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆aminoalkyl or a protecting group; and n is 1 or
 2. 2. The bicyclicnucleoside of claim 1 wherein Bx is an optionally protected pyrimidine,substituted pyrimidine, purine or substituted purine.
 3. The bicyclicnucleoside of claim 2 wherein Bx is uracil, thymine, cytosine,4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine,adenine, 6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine.
 4. Thebicyclic nucleoside of claim 1 wherein at least one of Q₁, Q₂, G₁ and G₂is CH₃ and the remaining of Q₁, Q₂, G₁ and G₂ are each H.
 5. Thebicyclic nucleoside of claim 1 wherein Q₁, Q₂, G₁ and G₂ are each H. 6.The bicyclic nucleoside of claim 1 wherein T₁ is 4,4′-dimethoxytrityland T₂ is diisopropylcyanoethoxy phosphoramidite.
 7. The bicyclicnucleoside of claim 1 wherein n is 1 and the configuration at the sulfuratom is R.
 8. The bicyclic nucleoside of claim 1 wherein n is 1 and theconfiguration at the sulfur atom is S.
 9. The bicyclic nucleoside ofclaim 1 wherein n is
 2. 10. The bicyclic nucleoside of claim 1 whereinQ₁, Q₂, G₁ and G₂ are each H, T₁ is 4,4′-dimethoxytrityl and T₂ isdiisopropylcyanoethoxy phosphoramidite.
 11. An oligomeric compoundcomprising at least one bicyclic nucleoside of Formula IIa:

wherein independently for each bicyclic nucleoside of Formula IIa: Bx isa heterocyclic base moiety; one of T₃ and T₄ is an internucleosidelinking group attaching the bicyclic nucleoside to the remainder of oneof the 5′ or 3′ end of the oligomeric compound and the other of T₃ andT₄ is hydroxyl, a protected hydroxyl, a 5′ or 3′ terminal group or aninternucleoside linking group attaching the bicyclic nucleoside to theremainder of the other of the 5′ or 3′ end of the oligomeric compound;Q₁ and Q₂ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl; G₁ and G₂ are each independently, H, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; each substitutedgroup is, independently, mono or poly substituted with substituentgroups independently selected from halogen, OJ₁, SJ₁, NJ₁J₂, N₃, CN,C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ andN(H)C(═S)NJ₁J₂; each J₁ and J₂ is, independently, H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ aminoalkyl or a protecting group; n is 1or 2; and wherein said oligomeric compound comprises from 8 to 40monomeric subunits linked by internucleoside linking groups and whereinat least some of the heterocyclic base moieties are capable ofhybridizing to a nucleic acid molecule.
 12. The oligomeric compound ofclaim 11 wherein each Bx is, independently, uracil, thymine, cytosine,4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine,adenine, 6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine.
 13. Theoligomeric compound of claim 11 comprising at least one 5′ or3′-conjugate group.
 14. The oligomeric compound of claim 11 wherein atleast one of Q₁, Q₂, G₁ and G₂ is CH₃ and the remaining of Q₁, Q₂, G₁and G₂ are each H for each bicyclic nucleoside of Formula IIa.
 15. Theoligomeric compound of claim 11 wherein Q₁, Q₂, G₁ and G₂ are each H foreach is bicyclic nucleoside of Formula IIa.
 16. The oligomeric compoundof claim 11 wherein n is 1 and the configuration at the sulfur atom is Rfor each bicyclic nucleoside of Formula IIa.
 17. The oligomeric compoundof claim 11 wherein n is 1 and the configuration at the sulfur atom is Sfor each bicyclic nucleoside of Formula IIa.
 18. The oligomeric compoundof claim 11 wherein n is 2 for each bicyclic nucleoside of Formula IIa.19. The oligomeric compound of claim 11 comprising from about 8 to about20 linked monomer subunits.
 20. The oligomeric compound of claim 11comprising a gapped oligomeric compound comprising a first regionconsisting of from 2 to 5 modified nucleosides, a second regionconsisting of from 2 to 5 modified nucleosides and a gap region locatedbetween the first and second regions, wherein at least one of themodified nucleosides of the first and second region is a bicyclicnucleoside having Formula IIa and wherein each monomer subunit in thegap region is independently, a nucleoside or a modified nucleoside thatis different from each bicyclic nucleoside of Formula IIa and each ofthe modified nucleosides in the first and second regions.
 21. Theoligomeric compound of claim 20 wherein the gap region comprises fromabout 8 to about 14 contiguous β-D-2′-deoxyribonucleosides.
 22. Theoligomeric compound of claim 20 wherein essentially each modifiednucleoside of the first and second region is a bicyclic nucleosidehaving Formula IIa.
 23. The oligomeric compound of claim 20 wherein eachof the modified nucleosides that is other than a bicyclic nucleosidehaving Formula II is, independently, a substituted nucleoside having oneor more sugar substituent groups, an optionally substituted 4′-Snucleoside or an optionally substituted bicyclic nucleoside.
 24. Theoligomeric compound of claim 11 wherein each internucleoside linkinggroup is, independently, a phosphodiester internucleoside linking groupor a phosphorothioate internucleoside linking group.
 25. The oligomericcompound of claim 11 wherein essentially each internucleoside linkinggroup is a phosphorothioate internucleoside linking group.
 26. A methodof inhibiting gene expression comprising contacting a cell with anoligomeric compound of claim 11 wherein said oligomeric compound iscomplementary to a target RNA.